November 2004
Volume 45, Issue 11
Jump To...
Free
Lens  |   November 2004
Chick δ1-Crystallin Enhancer Influences Mouse αA-Crystallin Promoter Activity in Transgenic Mice
Lixing W. Reneker; Qin Chen; Amy Bloch; Leike Xie; Gaby Schuster; Paul A. Overbeek
Author Affiliations
  • Lixing W. Reneker
    From the Department of Ophthalmology and the
  • Qin Chen
    Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, Texas.
  • Amy Bloch
    School of Medicine, University of Missouri, Columbia, Missouri; and the
  • Leike Xie
    From the Department of Ophthalmology and the
  • Gaby Schuster
    Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, Texas.
  • Paul A. Overbeek
    Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, Texas.
Investigative Ophthalmology & Visual Science November 2004, Vol.45, 4083-4090. doi:https://doi.org/10.1167/iovs.03-1270
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      User Alerts
      You are adding an alert for:
      Chick δ1-Crystallin Enhancer Influences Mouse αA-Crystallin Promoter Activity in Transgenic Mice
      You will receive an email whenever this article is corrected, updated, or cited in the literature. You can manage this and all other alerts in My Account
      The alert will be sent to:
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation
      Citation

      Lixing W. Reneker, Qin Chen, Amy Bloch, Leike Xie, Gaby Schuster, Paul A. Overbeek; Chick δ1-Crystallin Enhancer Influences Mouse αA-Crystallin Promoter Activity in Transgenic Mice. Invest. Ophthalmol. Vis. Sci. 2004;45(11):4083-4090. https://doi.org/10.1167/iovs.03-1270.

      Download citation file:

      © ARVO (1962-2015); The Authors (2016-present)

      ×
    • Get Permissions
  • Supplements
Abstract

purpose. Both the −366/+43 and the −282/+43 mouse αA-crystallin (or αA) promoters have been effective at driving transgene expression in lens fiber cells, but not in lens epithelium. Because the chick δ1-crystallin gene is expressed in lens epithelial cells, an enhancer was borrowed from this gene and linked to the αA promoter. This heterogenic enhancer/promoter construct was tested in transgenic mice to see whether it was active in both lens epithelium and fiber cells while retaining lens specificity.

methods. The third intron of the chick δ1-crystallin gene, which contains a lens enhancer element, was added to the 5′ end of the mouse αA promoter. We refer to this chimeric regulatory element as the δenαA promoter. To test its activity, we inserted coding sequences for five different genes. Transgenic mice were generated by pronuclear microinjection. Transgene expression patterns were analyzed by either X-gal staining, in situ hybridization or immunohistochemical staining.

results. When δenαA-lacZ transgenic embryos were stained with X-gal at embryonic day (E)11.5, β-galactosidase activity was detected only in the eye. Histologic sections of the stained embryos revealed that lacZ was expressed exclusively in the lens, in both epithelial and fiber cells. Transgenic mice were also generated using either the original αA- or the new δenαA promoter linked to an insulin cDNA. In situ hybridizations confirmed that the short αA promoter targeted prenatal insulin expression specifically to the lens fiber cells, whereas the δenαA promoter was active in both lens epithelial and fiber cells. Developmental studies of the δenαA-insulin mice showed that the δenαA promoter became active at the lens pit stage and remained active in all lens cells, even at postnatal ages. The δenαA promoter also successfully directed expression of SV40 T-antigen (TAg), human E2F2, and dominant negative Sprouty2 (dn-Spry2) genes to lens epithelial and fiber cells. The lens specificity of the δenαA promoter was maintained in minigenes with different types of introns and polyadenylation signals.

conclusions. A new lens-specific regulatory element was generated—the δenαA promoter, which can drive high levels of transgene expression in both lens epithelium and fiber cells throughout development. This modified promoter can be used for future transgenic studies of signal transduction and cell cycle regulation in lens epithelial cells.

The vertebrate ocular lens contains two cell types, epithelial and fiber cells, derived from the surface ectoderm. 1 2 3 The mouse αA-crystallin promoter has been used to obtain lens-specific gene expression in transgenic mice, thereby allowing in vivo modifications of lens differentiation and development. 4 5 6 7 For example, central lens epithelial cells can be induced to differentiate into lens fiber cells in transgenic mice by expression of secreted fibroblast growth factors (FGFs) under the control of the αA-crystallin promoter. 8 This promoter has been used in transgenic mice to express growth factors, 6 7 9 10 cell cycle regulators, 4 5 11 and transcription factors 12 that modify lens and eye development. 
Although the mouse αA-crystallin promoter is active in the lens, its activity in lens epithelial cells is much lower than its activity in lens fiber cells, based on in situ hybridization assays. 13 It would be useful to have a promoter with high activity in all lens cells. Such a promoter would be especially valuable for experiments designed to activate or inhibit intracellular signal-transduction pathways before the onset of fiber cell differentiation. The αB-crystallin promoter and the Pax6 surface ectodermal P0 promoter can direct transgene expression to lens epithelial and fiber cells. 14 15 However, transgene expression is not limited to the lens. 14 15 Because the αA-crystallin promoter is lens specific, we tested whether addition of an enhancer might expand its domain of activity. 
δ-Crystallin is a major lens protein in birds and reptiles. 16 The δ1-crystallin gene is expressed abundantly in the embryonic chick lens and is detected in the lens epithelium from the onset of lens differentiation. 17 This lens-specific expression is regulated by an enhancer element that is located in the third intron of the gene. 18 Although δ1-crystallin gene is not present in mammals, the cloned chick gene is expressed in a lens-specific manner in transgenic mice, 19 suggesting that the transregulatory proteins responsible for lens-specific expression of δ1-crystalllin are conserved across species. The chick δ1-crystallin enhancer, when linked to the Hsp68 promoter, can direct transgene (lacZ) expression in both lens epithelial and fiber cells in the transgenic mouse embryo. 20 It is not clear whether the δ1-crystallin enhancer is also capable of directing transgene expression in the postnatal mouse lens. 
Based on these previous studies, we tested the consequences of adding the δ1-crystallin enhancer to the 5′ end of the αA-crystallin promoter. This new chimeric promoter was termed δenαA. We constructed five different minigenes and tested them for expression in transgenic mice. Herein, we provide evidence that the δenαA promoter can drive transgene expression in both lens epithelial and fiber cells throughout development while remaining lens specific. 
Materials and Methods
Construction of Minigenes
A plasmid clone (pHspZδen) containing the chick δ1-crystallin lens enhancer element was generously provided by Hisato Kondoh (Osaka University, Osaka, Japan). 18 This enhancer is approximately 1 kb in length and spans the entire third intron of the δ1-crystallin gene. A KpnI-BglII fragment (1 kb) containing the enhancer was linked to a KpnI-BglII–digested mouse αA crystallin promoter vector plasmid. 21 22 The resultant plasmid was modified by deleting the EcoRI site in the αA-crystallin promoter. 
We constructed two promoter vectors for transgene expression (see Fig. 1 ). For vector 1, an adenovirus intron (minx) from Sue Berget (Baylor College of Medicine, Houston, TX) 23 and a 230-bp polyA signal from the mouse αB crystallin gene were used. The αB polyA sequences were amplified by polymerase chain reaction (PCR), using primers, 5′-TCCCCCGGGTAGATCCCCTTTCCTCATTG-3′ (sense) and 5′-GCTCAGACCCCTGAATCATAGTTTG-3′ (antisense) and then digested with XmaI and XbaI for cloning. For vector 2, the δenαA promoter was placed upstream from a rabbit β-globin intron and human growth hormone (hGH) polyA signal. 24 25  
The five minigenes were designed as follows: (1) To construct the lacZ reporter gene (see Fig. 2A ) in vector I, lacZ sequences were released from plasmid pKS-hsp-lacZ-pA obtained from Janet Rossant (Samuel Lunenfeld Research Institute of Mount Sinai Hospital, Toronto, Canada) by digestion with BstEII and XbaI, blunt ended and ligated into the EcoRV site of vector 1 (Fig. 1) . (2) The human insulin cDNA clone was obtained from Graeme Bell (University of Chicago, Chicago, IL). The insulin cDNA was released by EcoRI digestion and inserted into the EcoRI site of the CPV2 plasmid 6 to make the αA-insulin minigene (see Fig. 3A ), or blunt ended and inserted into the SmaI site of vector 2 to make the δenαA-insulin minigene (see Fig. 3B ). (3) The SV40 early region (approximately 2.7 kb) was released from the αA-cry-TAg plasmid 26 by BamHI/EcoRI digestion and inserted into a BamHI/EcoRI digest of vector 1 to make the δenαA-TAg minigene (see Fig. 6 ). (4) To construct δenαA-E2F2, the human cDNA was isolated from CPV2-E2F2 11 by digestion with EcoRI (blunt ended) and then ClaI. The fragment was inserted into a SmaI/ClaI digest of vector 1 (see Fig. 7 ). (5) The dn-Spry2 cDNA clone was provided by Eisuke Nishida (Kyoto University, Kyoto, Japan). 27 The cDNA fragment was cut out by BglII digestion, blunted with T4 DNA polymerase, and inserted into the SmaI site of vector 2 (see Fig. 8 ). 
Generation and Screening of Transgenic Mice
All animals were used in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Transgenic mice were generated by pronuclear injection of purified DNA fragments into one-cell-stage inbred FVB/N embryos. 28 Transgenic mice expressing the lacZ gene under the control of a 5-kb Pax6 promoter were obtained from Richard Lang (University of Cincinnati, Cincinnati, OH). 29  
Genomic DNAs from mouse tails or embryonic tissues were isolated and subjected to PCR screening according to procedures described previously. 13 For transgenic mice that were made with Pax6-lacZ (Fig. 2B) , CPV2-insulin (see Fig. 3A ), and δenαA-TAg (see Fig. 6 ), primers specific for SV40 sequences were used (SV40A, 5′-GTGAAGGAACCTTACTCTGTGGTG-3′; SV40B, 5′-GTCCTTGGGGTCTTCTACCTTTCTC-3′). 13 For transgenic mice that were made with vector 1, including δenαA-lacZ (see Fig. 2A ) and δenαA-E2F2 (see Fig. 7 ), we used primers homologous to the mouse αA-crystallin promoter and the minx intron. They are 5′-GCATTCCAGCTGCTGACGGT-3′ (Pr4, sense) and 5′-AGAGGATCCCCACTGGAAAGAC-3′ (minx, antisense). To screen the mice made with minigenes in vector 2, including δenαA-insulin (see Figs. 3B 4 5 ) and δenαA-dnSpry2 (see Fig. 8 ), we used Pr4 as the sense primer along with an antisense primer (β3, 5′-AAGGCATGAACATGGTTAGCAGAGG-3′) homologous to the rabbit β-globin intron sequences. 
Staining for β-Galactosidase Activity
Mouse embryos were isolated from timed-pregnant female mice and fixed in 2% paraformaldehyde plus 0.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) at 4°C for 1 hour. Embryos were rinsed three times (30 minute each) in washing solution containing 0.1 M phosphate buffer (pH 7.3), 2 mM MgCl2, 0.01% sodium deoxycholate, and 0.02% NP-40. Embryos were then stained at 4°C for 4 to 12 hours in X-gal staining solution (washing solution plus 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 1 mg/mL X-gal). Stained embryos were rinsed in phosphate-buffered saline (PBS; pH 7.4) and stored in 70% ethanol. They were subsequently cleared (Histo-Clear; National Diagnostics, Atlanta, GA) and embedded in paraffin for sectioning. Tissue sections were counterstained with nuclear fast red (PolyScientific, Bayshore, NY). 
In Situ Hybridization
We used in situ hybridizations to detect expression of the other transgenes. 30 For transgenes made in vector 2 (see Figs. 3B 4 5 8 ), we subcloned the human growth hormone (hGH) polyA sequences into the EcoRI site of a plasmid (Bluescript SK−; Stratagene, La Jolla, CA). The orientation of the hGH insert was determined by DNA sequencing. Sense and antisense riboprobes were generated from linearized DNA templates by in vitro transcription using T3 or T7 RNA polymerase (Promega, Madison, WI). For transgenic mice carrying CPV2-insulin (see Fig. 3A ) or δenαA-TAg (see Fig. 6B 6C 6D 6E ), a riboprobe specific to the SV40 sequences was made as previously described. 13 For the δenαA-E2F2 mice (see Fig. 7 ), the human E2F2 cDNA was subcloned into the plasmid pBluescript and used to generate riboprobes. 11 Tissues for analysis were fixed, embedded, sectioned, and then hybridized to 35S-labeled riboprobes. After the slides were washed and dried, they were dipped in emulsion (NTB-2; Eastman Kodak, Rochester, NY) and exposed at 4°C for 2 to 7 days before development (D-19 developer; Eastman Kodak). The slides were counterstained with Harris hematoxylin (PolyScientific). In situ hybridization signals were captured as bright- or dark-field images. 
Immunohistochemistry
Mouse tissues were fixed, processed, and sectioned for immunohistochemistry as previously described. 31 After pretreatment with 5% normal goat serum, tissue sections were incubated at 4°C overnight with a monoclonal antibody against SV40 T-antigen (TAg; BD Biosciences, Palo Alto, CA). Sections were rinsed with PBS and incubated with fluorescein-labeled anti-mouse IgG (Sigma-Aldrich, St. Louis, MO) at room temperature for 1 hour, to reveal the TAg localization. After they were rinsed with PBS, slides were mounted in PBS with 50% glycerol for examination and photography under blue-light illumination. 
Results
The endogenous mouse αA-crystallin gene is activated at embryonic day (E)10.5 in the lens vesicle and remains expressed postnatally. 32 A 400-bp mouse αA-crystallin (αA) promoter (−366/+43) has been used successfully to drive high levels of transgene expression in mouse lens. 4 13 33 One feature of the short promoter is that its activity is much higher in lens fiber cells than in lens epithelial cells in transgenic mice (see Fig. 3A , for example). For studies of cell cycle regulation and signal transduction in lens epithelial cells, it would be preferable to use a promoter that gives high levels of transgene expression in the lens epithelial cells. It has been reported that the proximal promoter (P0) of the Pax6 gene can drive transgene expression in both lens epithelial and fiber cells (Fig. 2B) . 15 34 However, this promoter is active in other ocular tissues including corneal, conjunctiva, and glandular epithelium (Fig. 2B) . 
δ-Crystallin is the most abundantly expressed crystallin in chick lens. 16 The third intron of the δ1-crystallin gene contains an enhancer element that determines the lens-specific expression of the gene. 18 In this study, we modified the mouse short αA promoter by adding the δ1-crystallin enhancer (δen) to the 5′ end of the promoter. We named the chimeric regulatory element the δenαA promoter. To test its activity in transgenic mice, we first constructed a lacZ reporter minigene (Fig. 2A) . The bacterial lacZ gene was flanked by a small intron from adenovirus (called minx) at its 5′ end and a polyA signal from the mouse αB-crystallin gene (αBpA) at its 3′ end. Two transgenic founder embryos were isolated at E11.5 and stained for β-galactosidase activity. Both embryos gave the staining pattern shown in Figure 2A . The eye was the only tissue that stained blue in the whole embryo (Fig. 2A , left). When the stained embryos were sectioned, X-gal staining was detected only in the lens (Fig. 2A , middle), and was present in both lens epithelial and fiber cells (Fig. 2A , right). 
To compare the specificity of the αA and δenαA promoters, they were linked to the same reporter gene, human insulin cDNA (Fig. 3) . We generated two transgenic lines with the αA-insulin minigene (CPV2-insulin). In both lines, in situ hybridization detected transgene expression only in the lens fiber cells (Fig. 3A) . Using the δenαA-insulin construct, six transgenic lines were established (LR12-14, -16, -20, and -22). In situ hybridizations performed on five of these lines showed transgene expression in both lens epithelial and fiber cells (Fig. 3B) . Different transgenic lines had different levels of transgene expression (Fig. 3B) . In general, the expression level was higher in the lens epithelial cells than in the fiber cells (except for line LR16). 
We also investigated the time course of δenαA promoter activity using three of the insulin transgenic lines (Figs. 4 5) . In the strong-expressing line LR22, we did not detect transgene expression in the lens placode but expression was detected readily in the cells of the lens pit (Figs. 4A 4B) . In other lines, transgene expression was detected in the late lens pit or early lens vesicle stage (Figs. 4C 4D 4E 4F ; see also Figs. 7A 7B 7C 7D ′, and Fig. 8A ). High levels of transgene expression were found in newborn (postnatal day [P]0), P7, and P30 lenses (Fig. 5) . Thus, the δenαA promoter remains active postnatally in the mouse, despite the fact that δ1-crystallin gene expression is turned off in chicks after hatching. 16  
We made additional minigenes using either vector 1 (Figs. 6 7) or vector 2 (Fig. 8) . For example, the δenαA promoter was linked to the SV40 early region, which encodes large TAg (Fig. 6) . This construct was used to establish four transgenic lines, OVE1664, -1765, -1766, and -1767. Mice from all four lines exhibited lens tumors. Tumors were not found in other parts of the body. For OVE1664, in situ hybridizations showed that the transgene is expressed in all lens cells at E15.5 (Figs. 6C 6E) . Protein expression in these cells was confirmed by fluorescent immunolabeling using an anti-TAg antibody (Fig. 6G) . 
For the E2F2 minigene (δenαA-minx-E2F2-αBpA; Fig. 7 ), two E15.5 transgenic founder embryos were isolated and tested for transgene expression by in situ hybridization using a probe homologous to the human E2F2 cDNA sequences. One founder embryo showed transgene expression in both lens epithelial and fiber cells, and the other had expression only in the fiber cells (data not shown). We also generated three stable transgenic lines (OVE1586, -1587, and -1588). Mice from line 1588 did not show any lens defects and transgene expression was not detected by reverse transcription (RT)-PCR analysis (data not shown). Cataracts developed in mice from lines OVE1586 and -1587. In situ hybridization showed that the transgene was expressed in both lens epithelial and fiber cells in line OVE1587 (Fig. 7) . At E10.5, a low level of transgene mRNA was detected in the lens pit. Transgene expression levels increased during prenatal development (Figs. 7D 7E 7F ′). In line OVE1586, E2F2 mRNA was detected only in lens fiber cells, by in situ hybridization (data not shown). The same pattern of expression occurred in CPV2-E2F2 transgenic mice. 11  
Recently, we generated nine transgenic founder mice with the δenαA-dnSpry2 minigene (Fig. 8) . The minigene was constructed using vector 2, as for the δenαA-insulin minigene (Fig. 3B) . Seven of the nine founder mice had cataracts with different degrees of severity. The founder mice were bred to the wild-type FVB/N mice to establish stable transgenic lines. In the severely affected lines (e.g., line LR40), a low level of transgene expression was first detected in the lens pit (Fig. 8A , E10.5) and then the expression level increased as lens development proceeded. At E12.5, high levels of transgene expression were found in both lens epithelial and fiber cells (Fig. 8A , E12.5), even though the expression level was still lower than that in most of the insulin families (Fig. 3B) . Transgene expression was also compared in the different dn-Spry2 lines (Fig. 8B) . In the low-expressing line (e.g., line LR44), transgene mRNA was not found in the lens pit but was detectable in the lens vesicle (data not shown). Among the three lines tested at E13.5, transgene expression was found in both lens epithelial and fiber cells. The levels are higher in lines LR37 and -40 than in line -44 (Fig. 8B) . 
In summary, we have now tested five minigenes in two different δenαA promoter vectors. Transgene expression was detected in both lens epithelial and fiber cells in 14 of the 16 integration sites that were assayed. Expression of the transgene begins at the lens pit stage of lens development or shortly thereafter. To date, expression has been detected only in the lens. No expression was seen in other regions of the eye or in other tissues of the embryos. 
Discussion
A short αA-crystallin promoter (−366/+43) in the mouse has been used previously to target transgene expression to the lens. 11 35 36 37 This promoter is significantly more active in lens fiber cells than in lens epithelial cells (see Fig. 3A , for example). In this study, we have added the chick δ1-crystallin enhancer to the short mouse αA-crystallin promoter. This new promoter, δenαA, was linked to four cDNAs (lacZ, insulin, E2F2 and dn-Spry2) and a viral gene SV40 T-antigen. These constructs were tested in transgenic mice. Each transgene was found to be expressed in a lens-specific fashion, often in both lens epithelial and fiber cells. In the δenαA-insulin mice, transgene expression was not found in the lens placode but was detected before closure of the lens pit during early lens development. Expression was still strong more than 4 weeks after birth. In the δenαA-dnSpry2 mice, a low level of transgene expression in the lens pit was detectable only in the high-expressing lines. The lens specificity of the δenαA promoter was retained with two different promoter vectors, 1 and 2 (Fig. 1) . We conclude that this novel chimeric promoter can be used to target transgene expression to all cells of the lens, beginning at the lens pit stage or shortly thereafter. A few transgenic families (2/16) had low or no expression in lens epithelial cells (e.g., the δenαA-E2F2 families), suggesting that some integration sites can inhibit the activity of the enhancer. 
δ1-Crystallin is abundantly expressed in the embryonic chick lens but is not present in the mammalian genome. 16 38 39 40 Nevertheless, the δ1-crystallin regulatory elements function in a lens-specific manner in transgenic mice. 19 41 This suggests that the trans-acting regulatory factors for δ1-crystallin expression are present in the mouse lens. Kondoh’s group has shown that Sox, Pax6, and Maf transcription factors act together to regulate δ1-crystallin enhancer activity. 20 42 43 Sox2 (or Sox1/Sox3) pairs with Pax6 to form an activation complex. Maf proteins augment the enhancer activity in fiber cells. Because the Sox, Pax6, and Maf genes are all expressed in the mouse lens during development, 44 45 46 47 48 it is likely that these transcription factors are recruited by the δ1-enhancer into the chimeric regulatory element, thereby promoting transgene expression in lens epithelial cells. Our results suggest that the short αA-crystallin promoter can be influenced by, or can respond to, transcriptional regulators that do not bind to the promoter directly. The short promoter is not active, or weakly active, in lens epithelial cells. However, it is capable of being activated in these cells. 
In our study, the δenαA promoter was active in the lens epithelium and fibers in 1-month-old postnatal lenses (Fig. 5) . This finding is intriguing because δ1-crystallin expression is turned off in the chick lens after hatching. Negative regulatory sites presumably exist in the chick δ1-crystallin gene to downregulate its expression after hatching. Such elements may not be contained within the 1-kb enhancer we used or, alternatively, may not bind to mouse proteins that can downregulate promoter activity. 
It is worthwhile to mention that in four of the five δenαA-insulin transgenic lines we assayed (LR12, -13, -14, and -22; Fig. 3B ), the transgene expression level was higher in the lens epithelial cells than in the fiber cells. It is known that insulin and insulin-like growth factors (IGFs) can stimulate chick, but not mouse, lens epithelial cells to differentiate into fiber cells. 49 50 51 In the δenαA-insulin transgenic mice, the lens epithelial cells did not undergo premature differentiation. However, we cannot rule out the possibility that insulin produced from the transgenic lens may enhance the δenαA chimeric promoter activity through a positive feedback mechanism. 
Pax6 plays a key role in eye and lens morphogenesis and development. There are three conserved transcription start sites (P0, P1, and α) in the mouse Pax6 locus. 15 Independent cis-regulatory elements have been identified, which control the tissue-specific patterns of expression of Pax6. 29 34 For example, Pax6 expression in the surface ectoderm is, at least in part, regulated by a highly conserved enhancer element located approximately 4 kb upstream of the P0 promoter in the mouse Pax6 gene. 29 34 This enhancer was shown to be sufficient to target expression of a lacZ reporter gene to the lens and corneal epithelium (Fig. 2B) . 34 Using a strategy and logic similar to the δ-enhancer experiments, we linked the Pax6 lens enhancer to the mouse αA-crystallin promoter. Transgenic mice were generated with the Pax6 enhancer-αA promoter linked to lacZ, FGF15, or p57/Kip2 (data not shown). The transgenic mice did not show consistent expression in lens epithelium and a significant fraction (almost 50%) showed no expression in the lens (data not shown). In contrast to the δ1-crystallin enhancer, the Pax6 enhancer did not cooperate effectively with the short αA-crystallin promoter. 
Although the endogenous αA-crystallin gene is expressed in lens epithelial cells, the short promoter has little or no activity in these cells. We presume that there are regulatory sequences used by the endogenous gene that are not included in the short promoter. These endogenous sequences have not been identified, but they may be located some distance from the short promoter. We also tested a longer promoter (1.3 kb) but did not see transgene expression in the epithelial cells (data not shown). The endogenous regulation of αA-crystallin expression may therefore involve communication and cooperation between the short promoter and more distant enhancer elements. The chick δ1-crystallin enhancer is apparently able to cooperate in an analogous way to induce the short promoter to be active in lens epithelial cells. The strategy of using an enhancer/promoter chimera to obtain stronger tissue-specific gene expression has been reported previously. 52 53 For example, a skeletal muscle gene (creatine kinase) enhancer was fused to the smooth muscle specific SM22α promoter to achieve stronger transgene expression in smooth muscle cells. 53 In our case, it is intriguing that the enhancer/promoter fusion changes the cell type specificity of the promoter but the promoter still remains tissue specific. 
In summary, we generated a new lens-specific promoter, δenαA, that can direct transgene expression to both prenatal and postnatal lens epithelial and fiber cells. Unlike the mouse αB-crystallin and Pax6 P0 promoters, the δenαA promoter is lens specific. Transgene expression was not detected in the cornea. This promoter should be particularly useful for transgenic studies of signal transduction and cell physiology in lens epithelial cells in vivo. It can also be used to express Cre and/or Flp recombinases to perform lens-specific gene deletion experiments in mice. 
 Supported by National Eye Institute Grants EY13146 (LWR) and EY10448 (PAO), core Grant EY14795, a departmental unrestricted grant from Research to Prevent Blindness Inc. (LWR), and a fellowship award from Knights Templar Eye Foundation (QC).
 Submitted for publication November 20, 2003; revised April 12 and July 11, 2004; accepted July 20, 2004.
 Disclosure: L.W. Reneker, None; Q. Chen, None; A. Bloch, None; L. Xie, None; G. Schuster, None; P.A. Overbeek, None
 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
 Corresponding author: Lixing W. Reneker, EC214 Mason Eye Institute, One Hospital Dr., Columbia, MO 65212; renekerl@health.missouri.edu.
 
Figure 1.
Diagram of δenαA promoter vectors. The chick δ1-crystallin enhancer (δen) was fused to a mouse short αA-crystallin promoter (αA) to make chimeric promoter δenαA. For vector 1, an adenovirus intron (minx) and a polyA signal from the mouse αB crystallin gene (αB pA, 230 bp) were used. The cDNA insertion site (insert) is indicated in each vector. For vector 2, a rabbit β-globin intron (640 bp) and human growth hormone polyA signal (hGH pA, 660 bp) were placed downstream from the δenαA promoter. Drawings are not to scale.
View OriginalDownload Slide
 
Diagram of δenαA promoter vectors. The chick δ1-crystallin enhancer (δen) was fused to a mouse short αA-crystallin promoter (αA) to make chimeric promoter δenαA. For vector 1, an adenovirus intron (minx) and a polyA signal from the mouse αB crystallin gene (αB pA, 230 bp) were used. The cDNA insertion site (insert) is indicated in each vector. For vector 2, a rabbit β-globin intron (640 bp) and human growth hormone polyA signal (hGH pA, 660 bp) were placed downstream from the δenαA promoter. Drawings are not to scale.
Figure 1.
 
Diagram of δenαA promoter vectors. The chick δ1-crystallin enhancer (δen) was fused to a mouse short αA-crystallin promoter (αA) to make chimeric promoter δenαA. For vector 1, an adenovirus intron (minx) and a polyA signal from the mouse αB crystallin gene (αB pA, 230 bp) were used. The cDNA insertion site (insert) is indicated in each vector. For vector 2, a rabbit β-globin intron (640 bp) and human growth hormone polyA signal (hGH pA, 660 bp) were placed downstream from the δenαA promoter. Drawings are not to scale.
Diagram of δenαA promoter vectors. The chick δ1-crystallin enhancer (δen) was fused to a mouse short αA-crystallin promoter (αA) to make chimeric promoter δenαA. For vector 1, an adenovirus intron (minx) and a polyA signal from the mouse αB crystallin gene (αB pA, 230 bp) were used. The cDNA insertion site (insert) is indicated in each vector. For vector 2, a rabbit β-globin intron (640 bp) and human growth hormone polyA signal (hGH pA, 660 bp) were placed downstream from the δenαA promoter. Drawings are not to scale.
View OriginalDownload Slide
Figure 2.
LacZ in transgenic mouse embryos. (A) δenαA-lacZ expression. Top: depiction of the δenαA-lacZ construct. A transgenic embryo (E11.5) stained with X-gal is shown. Blue (indicative of lacZ expression) was detected only in the eyes of the transgenic embryo (left, middle). After the stained embryo was sectioned, it was apparent that lacZ expression is lens specific (middle). A higher magnification shows that lacZ is expressed in both lens epithelial (right, L. epi., arrow) and fiber cells. (B) Pax6-lacZ expression. Pax6 P0 promoter-lacZ construct is shown at the top. SV40 small intron and polyA (pA) sequences are present at the 3′ end of the lacZ gene. A transgenic embryo (E13.5) stained with X-gal is shown. The transgene is expressed in the eye (blue, left). An embryo section shows lacZ activity (blue) in the lens as well as in the surface ectodermal tissues that later form the corneal and conjunctival epithelium (right, arrows).
View OriginalDownload Slide
 
LacZ in transgenic mouse embryos. (A) δenαA-lacZ expression. Top: depiction of the δenαA-lacZ construct. A transgenic embryo (E11.5) stained with X-gal is shown. Blue (indicative of lacZ expression) was detected only in the eyes of the transgenic embryo (left, middle). After the stained embryo was sectioned, it was apparent that lacZ expression is lens specific (middle). A higher magnification shows that lacZ is expressed in both lens epithelial (right, L. epi., arrow) and fiber cells. (B) Pax6-lacZ expression. Pax6 P0 promoter-lacZ construct is shown at the top. SV40 small intron and polyA (pA) sequences are present at the 3′ end of the lacZ gene. A transgenic embryo (E13.5) stained with X-gal is shown. The transgene is expressed in the eye (blue, left). An embryo section shows lacZ activity (blue) in the lens as well as in the surface ectodermal tissues that later form the corneal and conjunctival epithelium (right, arrows).
Figure 2.
 
LacZ in transgenic mouse embryos. (A) δenαA-lacZ expression. Top: depiction of the δenαA-lacZ construct. A transgenic embryo (E11.5) stained with X-gal is shown. Blue (indicative of lacZ expression) was detected only in the eyes of the transgenic embryo (left, middle). After the stained embryo was sectioned, it was apparent that lacZ expression is lens specific (middle). A higher magnification shows that lacZ is expressed in both lens epithelial (right, L. epi., arrow) and fiber cells. (B) Pax6-lacZ expression. Pax6 P0 promoter-lacZ construct is shown at the top. SV40 small intron and polyA (pA) sequences are present at the 3′ end of the lacZ gene. A transgenic embryo (E13.5) stained with X-gal is shown. The transgene is expressed in the eye (blue, left). An embryo section shows lacZ activity (blue) in the lens as well as in the surface ectodermal tissues that later form the corneal and conjunctival epithelium (right, arrows).
LacZ in transgenic mouse embryos. (A) δenαA-lacZ expression. Top: depiction of the δenαA-lacZ construct. A transgenic embryo (E11.5) stained with X-gal is shown. Blue (indicative of lacZ expression) was detected only in the eyes of the transgenic embryo (left, middle). After the stained embryo was sectioned, it was apparent that lacZ expression is lens specific (middle). A higher magnification shows that lacZ is expressed in both lens epithelial (right, L. epi., arrow) and fiber cells. (B) Pax6-lacZ expression. Pax6 P0 promoter-lacZ construct is shown at the top. SV40 small intron and polyA (pA) sequences are present at the 3′ end of the lacZ gene. A transgenic embryo (E13.5) stained with X-gal is shown. The transgene is expressed in the eye (blue, left). An embryo section shows lacZ activity (blue) in the lens as well as in the surface ectodermal tissues that later form the corneal and conjunctival epithelium (right, arrows).
View OriginalDownload Slide
Figure 3.
CPV2-insulin and δenαA-insulin expression in E15.5 lenses. (A) Human insulin (ins) cDNA was inserted into the CPV2 vector containing the mouse αA-crystallin promoter (αA). Transgene expression was detected by in situ hybridization with a 35S-labeled riboprobe homologous to the SV40 region of the minigene. Results from lines OVE441 (left) and -442 (right) are shown in bright-field images. Hybridization signal (black-silver grains) was detected only in the lens fiber cells. No significant transgene expression was detected in the lens epithelial cells (arrows). (B) Human insulin cDNA inserted into δenαA vector 2. The insulin (ins) coding sequences are flanked by an intron from the rabbit β-globin gene (β-globin) and a polyA signal from human growth hormone (hGHpA). In situ hybridization was performed with a 35S-labeled riboprobe homologous to the hGH sequences. A dark-field image shows that no signal was detected in the lens of a nontransgenic (NTG) mouse. Bright-field images illustrate the results in five different transgenic lines (LR12–14, -16, and -22). Transgene expression was lens specific in all lines and was detected in both lens epithelial and fiber cells. The epithelial cell expression for LR16 was relatively weak. Magnification of all images is the same. Scale bar, 100 μm.
View OriginalDownload Slide
 
CPV2-insulin and δenαA-insulin expression in E15.5 lenses. (A) Human insulin (ins) cDNA was inserted into the CPV2 vector containing the mouse αA-crystallin promoter (αA). Transgene expression was detected by in situ hybridization with a 35S-labeled riboprobe homologous to the SV40 region of the minigene. Results from lines OVE441 (left) and -442 (right) are shown in bright-field images. Hybridization signal (black-silver grains) was detected only in the lens fiber cells. No significant transgene expression was detected in the lens epithelial cells (arrows). (B) Human insulin cDNA inserted into δenαA vector 2. The insulin (ins) coding sequences are flanked by an intron from the rabbit β-globin gene (β-globin) and a polyA signal from human growth hormone (hGHpA). In situ hybridization was performed with a 35S-labeled riboprobe homologous to the hGH sequences. A dark-field image shows that no signal was detected in the lens of a nontransgenic (NTG) mouse. Bright-field images illustrate the results in five different transgenic lines (LR12–14, -16, and -22). Transgene expression was lens specific in all lines and was detected in both lens epithelial and fiber cells. The epithelial cell expression for LR16 was relatively weak. Magnification of all images is the same. Scale bar, 100 μm.
Figure 3.
 
CPV2-insulin and δenαA-insulin expression in E15.5 lenses. (A) Human insulin (ins) cDNA was inserted into the CPV2 vector containing the mouse αA-crystallin promoter (αA). Transgene expression was detected by in situ hybridization with a 35S-labeled riboprobe homologous to the SV40 region of the minigene. Results from lines OVE441 (left) and -442 (right) are shown in bright-field images. Hybridization signal (black-silver grains) was detected only in the lens fiber cells. No significant transgene expression was detected in the lens epithelial cells (arrows). (B) Human insulin cDNA inserted into δenαA vector 2. The insulin (ins) coding sequences are flanked by an intron from the rabbit β-globin gene (β-globin) and a polyA signal from human growth hormone (hGHpA). In situ hybridization was performed with a 35S-labeled riboprobe homologous to the hGH sequences. A dark-field image shows that no signal was detected in the lens of a nontransgenic (NTG) mouse. Bright-field images illustrate the results in five different transgenic lines (LR12–14, -16, and -22). Transgene expression was lens specific in all lines and was detected in both lens epithelial and fiber cells. The epithelial cell expression for LR16 was relatively weak. Magnification of all images is the same. Scale bar, 100 μm.
CPV2-insulin and δenαA-insulin expression in E15.5 lenses. (A) Human insulin (ins) cDNA was inserted into the CPV2 vector containing the mouse αA-crystallin promoter (αA). Transgene expression was detected by in situ hybridization with a 35S-labeled riboprobe homologous to the SV40 region of the minigene. Results from lines OVE441 (left) and -442 (right) are shown in bright-field images. Hybridization signal (black-silver grains) was detected only in the lens fiber cells. No significant transgene expression was detected in the lens epithelial cells (arrows). (B) Human insulin cDNA inserted into δenαA vector 2. The insulin (ins) coding sequences are flanked by an intron from the rabbit β-globin gene (β-globin) and a polyA signal from human growth hormone (hGHpA). In situ hybridization was performed with a 35S-labeled riboprobe homologous to the hGH sequences. A dark-field image shows that no signal was detected in the lens of a nontransgenic (NTG) mouse. Bright-field images illustrate the results in five different transgenic lines (LR12–14, -16, and -22). Transgene expression was lens specific in all lines and was detected in both lens epithelial and fiber cells. The epithelial cell expression for LR16 was relatively weak. Magnification of all images is the same. Scale bar, 100 μm.
View OriginalDownload Slide
Figure 4.
δenαA-insulin expression during early lens development. Embryos were isolated from lines LR22 (A, B) and -14 (C–F). 35S-labeled riboprobe homologous to the hGH region of the transgene (Fig. 3B) was used for in situ hybridization. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). In LR22, transgene expression was detected in the early lens pit (A, B). The transgene was similarly expressed in the cells of the lens pit (C, D) and lens vesicle (E, F) in line LR14. Scale bar, 100 μm.
View OriginalDownload Slide
 
δenαA-insulin expression during early lens development. Embryos were isolated from lines LR22 (A, B) and -14 (C–F). 35S-labeled riboprobe homologous to the hGH region of the transgene (Fig. 3B) was used for in situ hybridization. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). In LR22, transgene expression was detected in the early lens pit (A, B). The transgene was similarly expressed in the cells of the lens pit (C, D) and lens vesicle (E, F) in line LR14. Scale bar, 100 μm.
Figure 4.
 
δenαA-insulin expression during early lens development. Embryos were isolated from lines LR22 (A, B) and -14 (C–F). 35S-labeled riboprobe homologous to the hGH region of the transgene (Fig. 3B) was used for in situ hybridization. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). In LR22, transgene expression was detected in the early lens pit (A, B). The transgene was similarly expressed in the cells of the lens pit (C, D) and lens vesicle (E, F) in line LR14. Scale bar, 100 μm.
δenαA-insulin expression during early lens development. Embryos were isolated from lines LR22 (A, B) and -14 (C–F). 35S-labeled riboprobe homologous to the hGH region of the transgene (Fig. 3B) was used for in situ hybridization. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). In LR22, transgene expression was detected in the early lens pit (A, B). The transgene was similarly expressed in the cells of the lens pit (C, D) and lens vesicle (E, F) in line LR14. Scale bar, 100 μm.
View OriginalDownload Slide
Figure 5.
δenαA-insulin expression in postnatal lenses. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). Strong signals were found in both lens epithelial and fiber cells, from newborn (P0) to P30. The P30 transgenic lens had vacuolated regions, where fiber cells were degenerating. The transgenic lenses do not show premature differentiation of the lens epithelial cells. Scale bar, 100 μm.
View OriginalDownload Slide
 
δenαA-insulin expression in postnatal lenses. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). Strong signals were found in both lens epithelial and fiber cells, from newborn (P0) to P30. The P30 transgenic lens had vacuolated regions, where fiber cells were degenerating. The transgenic lenses do not show premature differentiation of the lens epithelial cells. Scale bar, 100 μm.
Figure 5.
 
δenαA-insulin expression in postnatal lenses. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). Strong signals were found in both lens epithelial and fiber cells, from newborn (P0) to P30. The P30 transgenic lens had vacuolated regions, where fiber cells were degenerating. The transgenic lenses do not show premature differentiation of the lens epithelial cells. Scale bar, 100 μm.
δenαA-insulin expression in postnatal lenses. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). Strong signals were found in both lens epithelial and fiber cells, from newborn (P0) to P30. The P30 transgenic lens had vacuolated regions, where fiber cells were degenerating. The transgenic lenses do not show premature differentiation of the lens epithelial cells. Scale bar, 100 μm.
View OriginalDownload Slide
Figure 6.
TAg expression at E15.5. The SV40 early region encoding large TAg was inserted into vector 1. (A) Expression of the transgene was assayed at E15.5 by in situ hybridization using a 35S-labeled SV40 riboprobe. The results are shown as bright-field (B, C) and dark-field (D, E) images. No signal was detected in the nontransgenic (NTG) lens (B, D). In line OVE1664, TAg was expressed in all the lens cells (C, E). Fluorescent immunohistochemical staining (F, G) showed that TAg protein is present in all cells of the transgenic lens.
View OriginalDownload Slide
 
TAg expression at E15.5. The SV40 early region encoding large TAg was inserted into vector 1. (A) Expression of the transgene was assayed at E15.5 by in situ hybridization using a 35S-labeled SV40 riboprobe. The results are shown as bright-field (B, C) and dark-field (D, E) images. No signal was detected in the nontransgenic (NTG) lens (B, D). In line OVE1664, TAg was expressed in all the lens cells (C, E). Fluorescent immunohistochemical staining (F, G) showed that TAg protein is present in all cells of the transgenic lens.
Figure 6.
 
TAg expression at E15.5. The SV40 early region encoding large TAg was inserted into vector 1. (A) Expression of the transgene was assayed at E15.5 by in situ hybridization using a 35S-labeled SV40 riboprobe. The results are shown as bright-field (B, C) and dark-field (D, E) images. No signal was detected in the nontransgenic (NTG) lens (B, D). In line OVE1664, TAg was expressed in all the lens cells (C, E). Fluorescent immunohistochemical staining (F, G) showed that TAg protein is present in all cells of the transgenic lens.
TAg expression at E15.5. The SV40 early region encoding large TAg was inserted into vector 1. (A) Expression of the transgene was assayed at E15.5 by in situ hybridization using a 35S-labeled SV40 riboprobe. The results are shown as bright-field (B, C) and dark-field (D, E) images. No signal was detected in the nontransgenic (NTG) lens (B, D). In line OVE1664, TAg was expressed in all the lens cells (C, E). Fluorescent immunohistochemical staining (F, G) showed that TAg protein is present in all cells of the transgenic lens.
View OriginalDownload Slide
Figure 7.
Expression of δenαA-E2F2. A 35S-labeled riboprobe for human E2F2 was used for in situ hybridizations. Bright-field images are shown in (A–F) and the corresponding dark-field images in (A′–F′). A low level of transgene expression was detected in the lens pit of the transgenic mouse at E10.5 (B, B′). By E12.5, transgene expression was detected in both lens epithelial and fiber cells (D, D′). All lens cells continued to express the transgene at E15.5 (F, F′). No signal was detected in the wild-type (WT) lenses. Scale bars, 50 μm.
View OriginalDownload Slide
 
Expression of δenαA-E2F2. A 35S-labeled riboprobe for human E2F2 was used for in situ hybridizations. Bright-field images are shown in (A–F) and the corresponding dark-field images in (A′–F′). A low level of transgene expression was detected in the lens pit of the transgenic mouse at E10.5 (B, B′). By E12.5, transgene expression was detected in both lens epithelial and fiber cells (D, D′). All lens cells continued to express the transgene at E15.5 (F, F′). No signal was detected in the wild-type (WT) lenses. Scale bars, 50 μm.
Figure 7.
 
Expression of δenαA-E2F2. A 35S-labeled riboprobe for human E2F2 was used for in situ hybridizations. Bright-field images are shown in (A–F) and the corresponding dark-field images in (A′–F′). A low level of transgene expression was detected in the lens pit of the transgenic mouse at E10.5 (B, B′). By E12.5, transgene expression was detected in both lens epithelial and fiber cells (D, D′). All lens cells continued to express the transgene at E15.5 (F, F′). No signal was detected in the wild-type (WT) lenses. Scale bars, 50 μm.
Expression of δenαA-E2F2. A 35S-labeled riboprobe for human E2F2 was used for in situ hybridizations. Bright-field images are shown in (A–F) and the corresponding dark-field images in (A′–F′). A low level of transgene expression was detected in the lens pit of the transgenic mouse at E10.5 (B, B′). By E12.5, transgene expression was detected in both lens epithelial and fiber cells (D, D′). All lens cells continued to express the transgene at E15.5 (F, F′). No signal was detected in the wild-type (WT) lenses. Scale bars, 50 μm.
View OriginalDownload Slide
Figure 8.
Expression of δenαA-dnSpry2 transgene. (A) Transgene expression in line LR40. A low level of transgene mRNA was first detected in the lens pit cells at E10.5. At E12.5 transgene expression was found at a high level in both lens epithelial and fiber cells. In situ hybridization signals are shown in both bright-field (left) and dark-field (right) images. (B) Transgene expression in different lines at E13.5. The expression was seen in both lens epithelial and fiber cells, and the levels were higher in lines LR37 and -40 than in line -44. The expression pattern is shown in dark-field images at low magnification (left) and bright-field images at high magnification (right). Scale bars, 50 μm.
View OriginalDownload Slide
 
Expression of δenαA-dnSpry2 transgene. (A) Transgene expression in line LR40. A low level of transgene mRNA was first detected in the lens pit cells at E10.5. At E12.5 transgene expression was found at a high level in both lens epithelial and fiber cells. In situ hybridization signals are shown in both bright-field (left) and dark-field (right) images. (B) Transgene expression in different lines at E13.5. The expression was seen in both lens epithelial and fiber cells, and the levels were higher in lines LR37 and -40 than in line -44. The expression pattern is shown in dark-field images at low magnification (left) and bright-field images at high magnification (right). Scale bars, 50 μm.
Figure 8.
 
Expression of δenαA-dnSpry2 transgene. (A) Transgene expression in line LR40. A low level of transgene mRNA was first detected in the lens pit cells at E10.5. At E12.5 transgene expression was found at a high level in both lens epithelial and fiber cells. In situ hybridization signals are shown in both bright-field (left) and dark-field (right) images. (B) Transgene expression in different lines at E13.5. The expression was seen in both lens epithelial and fiber cells, and the levels were higher in lines LR37 and -40 than in line -44. The expression pattern is shown in dark-field images at low magnification (left) and bright-field images at high magnification (right). Scale bars, 50 μm.
Expression of δenαA-dnSpry2 transgene. (A) Transgene expression in line LR40. A low level of transgene mRNA was first detected in the lens pit cells at E10.5. At E12.5 transgene expression was found at a high level in both lens epithelial and fiber cells. In situ hybridization signals are shown in both bright-field (left) and dark-field (right) images. (B) Transgene expression in different lines at E13.5. The expression was seen in both lens epithelial and fiber cells, and the levels were higher in lines LR37 and -40 than in line -44. The expression pattern is shown in dark-field images at low magnification (left) and bright-field images at high magnification (right). Scale bars, 50 μm.
View OriginalDownload Slide
The authors thank Dongcai Liang for generation of TAg mice; Lucy Chen for genotyping and maintaining the OVE1664 transgenic family; Larry Fromm and Shanavas Alikunju for making the CPV2-insulin and δenαA-lacZ minigene, respectively; Venkatesh Govindarajan, Lingkun Kong, and Barbara Harris for X-gal staining; Li Xu for constructing the δenαA-insulin minigene; Shan-yu Ho for genotyping the transgenic mice; the Transgenic Animal Core Facility at the University of Missouri-Columbia for generating the LR transgenic families; and Ales Cvekl at Albert Einstein College of Medicine for valuable and insightful discussions. 
Chow RL, Lang RA. Early eye development in vertebrates. Annu Rev Cell Dev Biol. 2001;17:255–296. [CrossRef] [PubMed]
McAvoy JW, Chamberlain CG, de Iongh RU, Hales AM, Lovicu FJ. Lens development. Eye. 1999;13:425–437. [CrossRef] [PubMed]
Jean D, Ewan K, Gruss P. Molecular regulators involved in vertebrate eye development. Mech Dev. 1998;76:3–18. [CrossRef] [PubMed]
Fromm L, Shawlot W, Gunning K, Butel JS, Overbeek PA. The retinoblastoma protein-binding region of simian virus 40 large T antigen alters cell cycle regulation in lenses of transgenic mice. Mol Cell Biol. 1994;14:6743–6754. [PubMed]
Pan H, Griep AE. Temporally distinct patterns of p53-dependent and p53-independent apoptosis during mouse lens development. Genes Dev. 1995;9:2157–2169. [CrossRef] [PubMed]
Reneker LW, Overbeek PA. Lens-specific expression of PDGF-A alters lens growth and development. Dev Biol. 1996;180:554–565. [CrossRef] [PubMed]
Shirke S, Faber SC, Hallem E, et al. Misexpression of IGF-I in the mouse lens expands the transitional zone and perturbs lens polarization. Mech Dev. 2001;101:167–174. [CrossRef] [PubMed]
Robinson ML, Ohtaka-Maruyama C, Chan CC, et al. Disregulation of ocular morphogenesis by lens-specific expression of FGF-3/int-2 in transgenic mice. Dev Biol. 1998;198:13–31. [CrossRef] [PubMed]
Srinivasan Y, Lovicu FJ, Overbeek PA. Lens-specific expression of transforming growth factor beta1 in transgenic mice causes anterior subcapsular cataracts. J Clin Invest. 1998;101:625–634. [CrossRef] [PubMed]
Lovicu FJ, Overbeek PA. Overlapping effects of different members of the FGF family on lens fiber differentiation in transgenic mice. Development. 1998;125:3365–3377. [PubMed]
Chen Q, Hung FC, Fromm L, Overbeek PA. Induction of cell cycle entry and cell death in postmitotic lens fiber cells by overexpression of E2F1 or E2F2. Invest Ophthalmol Vis Sci. 2000;41:4223–4231. [PubMed]
Goudreau G, Petrou P, Reneker LW, Graw J, Loster J, Gruss P. Mutually regulated expression of Pax6 and Six3 and its implications for the Pax6 haploinsufficient lens phenotype. Proc Natl Acad Sci USA. 2002;99:8719–8724. [CrossRef] [PubMed]
Reneker LW, Silversides DW, Patel K, Overbeek PA. TGF alpha can act as a chemoattractant to perioptic mesenchymal cells in developing mouse eyes. Development. 1995;121:1669–1680. [PubMed]
Gopal-Srivastava R, Kays WT, Piatigorsky J. Enhancer-independent promoter activity of the mouse alphaB-crystallin/small heat shock protein gene in the lens and cornea of transgenic mice. Mech Dev. 2000;92:125–134. [CrossRef] [PubMed]
Kammandel B, Chowdhury K, Stoykova A, Aparicio S, Brenner S, Gruss P. Distinct cis-essential modules direct the time-space pattern of the Pax6 gene activity. Dev Biol. 1999;205:79–97. [CrossRef] [PubMed]
Piatigorsky J. Delta crystallins and their nucleic acids. Mol Cell Biochem. 1984;59:33–56. [PubMed]
Piatigorsky J. Lens differentiation in vertebrates: a review of cellular and molecular features. Differentiation. 1981;19:134–153. [CrossRef] [PubMed]
Hayashi S, Goto K, Okada TS, Kondoh H. Lens-specific enhancer in the third intron regulates expression of the chicken delta 1-crystallin gene. Genes Dev. 1987;1:818–828. [CrossRef] [PubMed]
Takahashi Y, Hanaoka K, Hayasaka M, et al. Embryonic stem cell-mediated transfer and correct regulation of the chicken delta-crystallin gene in developing mouse embryos. Development. 1988;102:259–269. [PubMed]
Muta M, Kamachi Y, Yoshimoto A, Higashi Y, Kondoh H. Distinct roles of SOX2, Pax6 and Maf transcription factors in the regulation of lens-specific delta1-crystallin enhancer. Genes Cells. 2002;7:791–805. [CrossRef] [PubMed]
Chepelinsky AB, King CR, Zelenka PS, Piatigorsky J. Lens-specific expression of the chloramphenicol acetyltransferase gene promoted by 5′ flanking sequences of the murine alpha A-crystallin gene in explanted chicken lens epithelia. Proc Natl Acad Sci USA. 1985;82:2334–2338. [CrossRef] [PubMed]
Overbeek PA, Chepelinsky AB, Khillan JS, Piatigorsky J, Westphal H. Lens-specific expression and developmental regulation of the bacterial chloramphenicol acetyltransferase gene driven by the murine alpha A-crystallin promoter in transgenic mice. Proc Natl Acad Sci USA. 1985;82:7815–7819. [CrossRef] [PubMed]
Niwa M, Berget SM. Mutation of the AAUAAA polyadenylation signal depresses in vitro splicing of proximal but not distal introns. Genes Dev. 1991;5:2086–2095. [CrossRef] [PubMed]
Werner S, Weinberg W, Liao X, et al. Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation. EMBO J. 1993;12:2635–2643. [PubMed]
Robinson ML, MacMillan-Crow LA, Thompson JA, Overbeek PA. Expression of a truncated FGF receptor results in defective lens development in transgenic mice. Development. 1995;121:3959–3967. [PubMed]
Mahon KA, Chepelinsky AB, Khillan JS, Overbeek PA, Piatigorsky J, Westphal H. Oncogenesis of the lens in transgenic mice. Science. 1987;235:1622–1628. [CrossRef] [PubMed]
Hanafusa H, Torii S, Yasunaga T, Nishida E. Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway. Nature Cell Biol. 2002.850–858.
Taketo M, Schroeder AC, Mobraaten LE, et al. FVB/N: an inbred mouse strain preferable for transgenic analyses. Proc Natl Acad Sci USA. 1991;88:2065–2069. [CrossRef] [PubMed]
Dimanlig PV, Faber SC, Auerbach W, Makarenkova HP, Lang RA. The upstream ectoderm enhancer in Pax6 has an important role in lens induction. Development. 2001;128:4415–4424. [PubMed]
Xu L, Overbeek PA, Reneker LW. Systematic analysis of E-, N- and P-cadherin expression in mouse eye development. Exp Eye Res. 2002;74:753–760. [CrossRef] [PubMed]
Zhao S, Chen Q, Hung FC, Overbeek PA. BMP signaling is required for development of the ciliary body. Development. 2002;129:4435–4442. [PubMed]
Robinson ML, Overbeek PA. Differential expression of alpha A- and alpha B-crystallin during murine ocular development. Invest Ophthalmol Vis Sci. 1996;37:2276–2284. [PubMed]
Robinson ML, Overbeek PA, Verran DJ, et al. Extracellular FGF-1 acts as a lens differentiation factor in transgenic mice. Development. 1995;121:505–514. [PubMed]
Williams SC, Altmann CR, Chow RL, Hemmati-Brivanlou A, Lang RA. A highly conserved lens transcriptional control element from the Pax-6 gene. Mech Dev. 1998;73:225–229. [CrossRef] [PubMed]
Reneker LW, Overbeek PA. Lens-specific expression of PDGF-A in transgenic mice results in retinal astrocytic hamartomas. Invest Ophthalmol Vis Sci. 1996;37:2455–2466. [PubMed]
Stolen CM, Jackson MW, Griep AE. Overexpression of FGF-2 modulates fiber cell differentiation and survival in the mouse lens. Development. 1997;124:4009–4017. [PubMed]
Duncan MK, Cvekl A, Li X, Piatigorsky J. Truncated forms of Pax-6 disrupt lens morphology in transgenic mice. Invest Ophthalmol Vis Sci. 2000;41:464–473. [PubMed]
Nickerson JM, Piatigorsky J. Sequence of a complete chicken delta-crystallin cDNA. Proc Natl Acad Sci USA. 1984;81:2611–2615. [CrossRef] [PubMed]
Nickerson JM, Wawrousek EF, Hawkins JW, et al. The complete sequence of the chicken delta 1 crystallin gene and its 5′ flanking region. J Biol Chem. 1985;260:9100–9105. [PubMed]
Borras T, Nickerson JM, Chepelinsky AB, Piatigorsky J. Structural and functional evidence for differential promoter activity of the two linked delta-crystallin genes in the chicken. EMBO J. 1985;4:445–452. [PubMed]
Kondoh H, Katoh K, Takahashi Y, et al. Specific expression of the chicken delta-crystallin gene in the lens and the pyramidal neurons of the piriform cortex in transgenic mice. Dev Biol. 1987;120:177–185. [CrossRef] [PubMed]
Kamachi Y, Sockanathan S, Liu Q, Breitman M, Lovell-Badge R, Kondoh H. Involvement of SOX proteins in lens-specific activation of crystallin genes. EMBO J. 1995;14:3510–3519. [PubMed]
Kamachi Y, Uchikawa M, Tanouchi A, Sekido R, Kondoh H. Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development. Genes Dev. 2001;15:1272–1286. [CrossRef] [PubMed]
Cvekl A, Piatigorsky J. Lens development and crystallin gene expression: many roles for Pax-6. Bioessays. 1996;18:621–630. [CrossRef] [PubMed]
Collignon J, Sockanathan S, Hacker A, et al. A comparison of the properties of Sox-3 with Sry and two related genes, Sox-1 and Sox-2. Development. 1996;122:509–520. [PubMed]
Nishiguchi S, Wood H, Kondoh H, Lovell-Badge R, Episkopou V. Sox1 directly regulates the gamma-crystallin genes and is essential for lens development in mice. Genes Dev. 1998;12:776–781. [CrossRef] [PubMed]
Kawauchi S, Takahashi S, Nakajima O, et al. Regulation of lens fiber cell differentiation by transcription factor c-Maf. J Biol Chem. 1999;274:19254–19260. [CrossRef] [PubMed]
Ring BZ, Cordes SP, Overbeek PA, Barsh GS. Regulation of mouse lens fiber cell development and differentiation by the Maf gene. Development. 2000;127:307–317. [PubMed]
Beebe DC, Silver MH, Belcher KS, Van Wyk JJ, Svoboda ME, Zelenka PS. Lentropin, a protein that controls lens fiber formation, is related functionally and immunologically to the insulin-like growth factors. Proc Natl Acad Sci USA. 1987;84:2327–2330. [CrossRef] [PubMed]
Liu J, Chamberlain CG, McAvoy JW. IGF enhancement of FGF-induced fibre differentiation and DNA synthesis in lens explants. Exp Eye Res. 1996;63:621–629. [CrossRef] [PubMed]
Leenders WPJ, Vangenesen ST, Schoenmakers JGG, Vanzoelen EJJ, Lubsen NH. Synergism between temporally distinct growth factors: Bfgf, insulin and lens cell differentiation. Mech Dev. 1997;67:193–201. [CrossRef] [PubMed]
Gehrke S, Jerome V, Muller R. Chimeric transcriptional control units for improved liver-specific transgene expression. Gene. 2003;322:137–143. [CrossRef] [PubMed]
Ribault S, Neuville P, Mechine-Neuville A, et al. Chimeric smooth muscle-specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy. Circ Res. 2001;88:468–475. [CrossRef] [PubMed]
Figure 1.
Diagram of δenαA promoter vectors. The chick δ1-crystallin enhancer (δen) was fused to a mouse short αA-crystallin promoter (αA) to make chimeric promoter δenαA. For vector 1, an adenovirus intron (minx) and a polyA signal from the mouse αB crystallin gene (αB pA, 230 bp) were used. The cDNA insertion site (insert) is indicated in each vector. For vector 2, a rabbit β-globin intron (640 bp) and human growth hormone polyA signal (hGH pA, 660 bp) were placed downstream from the δenαA promoter. Drawings are not to scale.
View OriginalDownload Slide
 
Diagram of δenαA promoter vectors. The chick δ1-crystallin enhancer (δen) was fused to a mouse short αA-crystallin promoter (αA) to make chimeric promoter δenαA. For vector 1, an adenovirus intron (minx) and a polyA signal from the mouse αB crystallin gene (αB pA, 230 bp) were used. The cDNA insertion site (insert) is indicated in each vector. For vector 2, a rabbit β-globin intron (640 bp) and human growth hormone polyA signal (hGH pA, 660 bp) were placed downstream from the δenαA promoter. Drawings are not to scale.
Figure 1.
 
Diagram of δenαA promoter vectors. The chick δ1-crystallin enhancer (δen) was fused to a mouse short αA-crystallin promoter (αA) to make chimeric promoter δenαA. For vector 1, an adenovirus intron (minx) and a polyA signal from the mouse αB crystallin gene (αB pA, 230 bp) were used. The cDNA insertion site (insert) is indicated in each vector. For vector 2, a rabbit β-globin intron (640 bp) and human growth hormone polyA signal (hGH pA, 660 bp) were placed downstream from the δenαA promoter. Drawings are not to scale.
Diagram of δenαA promoter vectors. The chick δ1-crystallin enhancer (δen) was fused to a mouse short αA-crystallin promoter (αA) to make chimeric promoter δenαA. For vector 1, an adenovirus intron (minx) and a polyA signal from the mouse αB crystallin gene (αB pA, 230 bp) were used. The cDNA insertion site (insert) is indicated in each vector. For vector 2, a rabbit β-globin intron (640 bp) and human growth hormone polyA signal (hGH pA, 660 bp) were placed downstream from the δenαA promoter. Drawings are not to scale.
View OriginalDownload Slide
Figure 2.
LacZ in transgenic mouse embryos. (A) δenαA-lacZ expression. Top: depiction of the δenαA-lacZ construct. A transgenic embryo (E11.5) stained with X-gal is shown. Blue (indicative of lacZ expression) was detected only in the eyes of the transgenic embryo (left, middle). After the stained embryo was sectioned, it was apparent that lacZ expression is lens specific (middle). A higher magnification shows that lacZ is expressed in both lens epithelial (right, L. epi., arrow) and fiber cells. (B) Pax6-lacZ expression. Pax6 P0 promoter-lacZ construct is shown at the top. SV40 small intron and polyA (pA) sequences are present at the 3′ end of the lacZ gene. A transgenic embryo (E13.5) stained with X-gal is shown. The transgene is expressed in the eye (blue, left). An embryo section shows lacZ activity (blue) in the lens as well as in the surface ectodermal tissues that later form the corneal and conjunctival epithelium (right, arrows).
View OriginalDownload Slide
 
LacZ in transgenic mouse embryos. (A) δenαA-lacZ expression. Top: depiction of the δenαA-lacZ construct. A transgenic embryo (E11.5) stained with X-gal is shown. Blue (indicative of lacZ expression) was detected only in the eyes of the transgenic embryo (left, middle). After the stained embryo was sectioned, it was apparent that lacZ expression is lens specific (middle). A higher magnification shows that lacZ is expressed in both lens epithelial (right, L. epi., arrow) and fiber cells. (B) Pax6-lacZ expression. Pax6 P0 promoter-lacZ construct is shown at the top. SV40 small intron and polyA (pA) sequences are present at the 3′ end of the lacZ gene. A transgenic embryo (E13.5) stained with X-gal is shown. The transgene is expressed in the eye (blue, left). An embryo section shows lacZ activity (blue) in the lens as well as in the surface ectodermal tissues that later form the corneal and conjunctival epithelium (right, arrows).
Figure 2.
 
LacZ in transgenic mouse embryos. (A) δenαA-lacZ expression. Top: depiction of the δenαA-lacZ construct. A transgenic embryo (E11.5) stained with X-gal is shown. Blue (indicative of lacZ expression) was detected only in the eyes of the transgenic embryo (left, middle). After the stained embryo was sectioned, it was apparent that lacZ expression is lens specific (middle). A higher magnification shows that lacZ is expressed in both lens epithelial (right, L. epi., arrow) and fiber cells. (B) Pax6-lacZ expression. Pax6 P0 promoter-lacZ construct is shown at the top. SV40 small intron and polyA (pA) sequences are present at the 3′ end of the lacZ gene. A transgenic embryo (E13.5) stained with X-gal is shown. The transgene is expressed in the eye (blue, left). An embryo section shows lacZ activity (blue) in the lens as well as in the surface ectodermal tissues that later form the corneal and conjunctival epithelium (right, arrows).
LacZ in transgenic mouse embryos. (A) δenαA-lacZ expression. Top: depiction of the δenαA-lacZ construct. A transgenic embryo (E11.5) stained with X-gal is shown. Blue (indicative of lacZ expression) was detected only in the eyes of the transgenic embryo (left, middle). After the stained embryo was sectioned, it was apparent that lacZ expression is lens specific (middle). A higher magnification shows that lacZ is expressed in both lens epithelial (right, L. epi., arrow) and fiber cells. (B) Pax6-lacZ expression. Pax6 P0 promoter-lacZ construct is shown at the top. SV40 small intron and polyA (pA) sequences are present at the 3′ end of the lacZ gene. A transgenic embryo (E13.5) stained with X-gal is shown. The transgene is expressed in the eye (blue, left). An embryo section shows lacZ activity (blue) in the lens as well as in the surface ectodermal tissues that later form the corneal and conjunctival epithelium (right, arrows).
View OriginalDownload Slide
Figure 3.
CPV2-insulin and δenαA-insulin expression in E15.5 lenses. (A) Human insulin (ins) cDNA was inserted into the CPV2 vector containing the mouse αA-crystallin promoter (αA). Transgene expression was detected by in situ hybridization with a 35S-labeled riboprobe homologous to the SV40 region of the minigene. Results from lines OVE441 (left) and -442 (right) are shown in bright-field images. Hybridization signal (black-silver grains) was detected only in the lens fiber cells. No significant transgene expression was detected in the lens epithelial cells (arrows). (B) Human insulin cDNA inserted into δenαA vector 2. The insulin (ins) coding sequences are flanked by an intron from the rabbit β-globin gene (β-globin) and a polyA signal from human growth hormone (hGHpA). In situ hybridization was performed with a 35S-labeled riboprobe homologous to the hGH sequences. A dark-field image shows that no signal was detected in the lens of a nontransgenic (NTG) mouse. Bright-field images illustrate the results in five different transgenic lines (LR12–14, -16, and -22). Transgene expression was lens specific in all lines and was detected in both lens epithelial and fiber cells. The epithelial cell expression for LR16 was relatively weak. Magnification of all images is the same. Scale bar, 100 μm.
View OriginalDownload Slide
 
CPV2-insulin and δenαA-insulin expression in E15.5 lenses. (A) Human insulin (ins) cDNA was inserted into the CPV2 vector containing the mouse αA-crystallin promoter (αA). Transgene expression was detected by in situ hybridization with a 35S-labeled riboprobe homologous to the SV40 region of the minigene. Results from lines OVE441 (left) and -442 (right) are shown in bright-field images. Hybridization signal (black-silver grains) was detected only in the lens fiber cells. No significant transgene expression was detected in the lens epithelial cells (arrows). (B) Human insulin cDNA inserted into δenαA vector 2. The insulin (ins) coding sequences are flanked by an intron from the rabbit β-globin gene (β-globin) and a polyA signal from human growth hormone (hGHpA). In situ hybridization was performed with a 35S-labeled riboprobe homologous to the hGH sequences. A dark-field image shows that no signal was detected in the lens of a nontransgenic (NTG) mouse. Bright-field images illustrate the results in five different transgenic lines (LR12–14, -16, and -22). Transgene expression was lens specific in all lines and was detected in both lens epithelial and fiber cells. The epithelial cell expression for LR16 was relatively weak. Magnification of all images is the same. Scale bar, 100 μm.
Figure 3.
 
CPV2-insulin and δenαA-insulin expression in E15.5 lenses. (A) Human insulin (ins) cDNA was inserted into the CPV2 vector containing the mouse αA-crystallin promoter (αA). Transgene expression was detected by in situ hybridization with a 35S-labeled riboprobe homologous to the SV40 region of the minigene. Results from lines OVE441 (left) and -442 (right) are shown in bright-field images. Hybridization signal (black-silver grains) was detected only in the lens fiber cells. No significant transgene expression was detected in the lens epithelial cells (arrows). (B) Human insulin cDNA inserted into δenαA vector 2. The insulin (ins) coding sequences are flanked by an intron from the rabbit β-globin gene (β-globin) and a polyA signal from human growth hormone (hGHpA). In situ hybridization was performed with a 35S-labeled riboprobe homologous to the hGH sequences. A dark-field image shows that no signal was detected in the lens of a nontransgenic (NTG) mouse. Bright-field images illustrate the results in five different transgenic lines (LR12–14, -16, and -22). Transgene expression was lens specific in all lines and was detected in both lens epithelial and fiber cells. The epithelial cell expression for LR16 was relatively weak. Magnification of all images is the same. Scale bar, 100 μm.
CPV2-insulin and δenαA-insulin expression in E15.5 lenses. (A) Human insulin (ins) cDNA was inserted into the CPV2 vector containing the mouse αA-crystallin promoter (αA). Transgene expression was detected by in situ hybridization with a 35S-labeled riboprobe homologous to the SV40 region of the minigene. Results from lines OVE441 (left) and -442 (right) are shown in bright-field images. Hybridization signal (black-silver grains) was detected only in the lens fiber cells. No significant transgene expression was detected in the lens epithelial cells (arrows). (B) Human insulin cDNA inserted into δenαA vector 2. The insulin (ins) coding sequences are flanked by an intron from the rabbit β-globin gene (β-globin) and a polyA signal from human growth hormone (hGHpA). In situ hybridization was performed with a 35S-labeled riboprobe homologous to the hGH sequences. A dark-field image shows that no signal was detected in the lens of a nontransgenic (NTG) mouse. Bright-field images illustrate the results in five different transgenic lines (LR12–14, -16, and -22). Transgene expression was lens specific in all lines and was detected in both lens epithelial and fiber cells. The epithelial cell expression for LR16 was relatively weak. Magnification of all images is the same. Scale bar, 100 μm.
View OriginalDownload Slide
Figure 4.
δenαA-insulin expression during early lens development. Embryos were isolated from lines LR22 (A, B) and -14 (C–F). 35S-labeled riboprobe homologous to the hGH region of the transgene (Fig. 3B) was used for in situ hybridization. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). In LR22, transgene expression was detected in the early lens pit (A, B). The transgene was similarly expressed in the cells of the lens pit (C, D) and lens vesicle (E, F) in line LR14. Scale bar, 100 μm.
View OriginalDownload Slide
 
δenαA-insulin expression during early lens development. Embryos were isolated from lines LR22 (A, B) and -14 (C–F). 35S-labeled riboprobe homologous to the hGH region of the transgene (Fig. 3B) was used for in situ hybridization. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). In LR22, transgene expression was detected in the early lens pit (A, B). The transgene was similarly expressed in the cells of the lens pit (C, D) and lens vesicle (E, F) in line LR14. Scale bar, 100 μm.
Figure 4.
 
δenαA-insulin expression during early lens development. Embryos were isolated from lines LR22 (A, B) and -14 (C–F). 35S-labeled riboprobe homologous to the hGH region of the transgene (Fig. 3B) was used for in situ hybridization. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). In LR22, transgene expression was detected in the early lens pit (A, B). The transgene was similarly expressed in the cells of the lens pit (C, D) and lens vesicle (E, F) in line LR14. Scale bar, 100 μm.
δenαA-insulin expression during early lens development. Embryos were isolated from lines LR22 (A, B) and -14 (C–F). 35S-labeled riboprobe homologous to the hGH region of the transgene (Fig. 3B) was used for in situ hybridization. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). In LR22, transgene expression was detected in the early lens pit (A, B). The transgene was similarly expressed in the cells of the lens pit (C, D) and lens vesicle (E, F) in line LR14. Scale bar, 100 μm.
View OriginalDownload Slide
Figure 5.
δenαA-insulin expression in postnatal lenses. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). Strong signals were found in both lens epithelial and fiber cells, from newborn (P0) to P30. The P30 transgenic lens had vacuolated regions, where fiber cells were degenerating. The transgenic lenses do not show premature differentiation of the lens epithelial cells. Scale bar, 100 μm.
View OriginalDownload Slide
 
δenαA-insulin expression in postnatal lenses. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). Strong signals were found in both lens epithelial and fiber cells, from newborn (P0) to P30. The P30 transgenic lens had vacuolated regions, where fiber cells were degenerating. The transgenic lenses do not show premature differentiation of the lens epithelial cells. Scale bar, 100 μm.
Figure 5.
 
δenαA-insulin expression in postnatal lenses. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). Strong signals were found in both lens epithelial and fiber cells, from newborn (P0) to P30. The P30 transgenic lens had vacuolated regions, where fiber cells were degenerating. The transgenic lenses do not show premature differentiation of the lens epithelial cells. Scale bar, 100 μm.
δenαA-insulin expression in postnatal lenses. Bright-field images are shown in (A), (C), and (E), and their matching dark-field images in (B), (D), and (F). Strong signals were found in both lens epithelial and fiber cells, from newborn (P0) to P30. The P30 transgenic lens had vacuolated regions, where fiber cells were degenerating. The transgenic lenses do not show premature differentiation of the lens epithelial cells. Scale bar, 100 μm.
View OriginalDownload Slide
Figure 6.
TAg expression at E15.5. The SV40 early region encoding large TAg was inserted into vector 1. (A) Expression of the transgene was assayed at E15.5 by in situ hybridization using a 35S-labeled SV40 riboprobe. The results are shown as bright-field (B, C) and dark-field (D, E) images. No signal was detected in the nontransgenic (NTG) lens (B, D). In line OVE1664, TAg was expressed in all the lens cells (C, E). Fluorescent immunohistochemical staining (F, G) showed that TAg protein is present in all cells of the transgenic lens.
View OriginalDownload Slide
 
TAg expression at E15.5. The SV40 early region encoding large TAg was inserted into vector 1. (A) Expression of the transgene was assayed at E15.5 by in situ hybridization using a 35S-labeled SV40 riboprobe. The results are shown as bright-field (B, C) and dark-field (D, E) images. No signal was detected in the nontransgenic (NTG) lens (B, D). In line OVE1664, TAg was expressed in all the lens cells (C, E). Fluorescent immunohistochemical staining (F, G) showed that TAg protein is present in all cells of the transgenic lens.
Figure 6.
 
TAg expression at E15.5. The SV40 early region encoding large TAg was inserted into vector 1. (A) Expression of the transgene was assayed at E15.5 by in situ hybridization using a 35S-labeled SV40 riboprobe. The results are shown as bright-field (B, C) and dark-field (D, E) images. No signal was detected in the nontransgenic (NTG) lens (B, D). In line OVE1664, TAg was expressed in all the lens cells (C, E). Fluorescent immunohistochemical staining (F, G) showed that TAg protein is present in all cells of the transgenic lens.
TAg expression at E15.5. The SV40 early region encoding large TAg was inserted into vector 1. (A) Expression of the transgene was assayed at E15.5 by in situ hybridization using a 35S-labeled SV40 riboprobe. The results are shown as bright-field (B, C) and dark-field (D, E) images. No signal was detected in the nontransgenic (NTG) lens (B, D). In line OVE1664, TAg was expressed in all the lens cells (C, E). Fluorescent immunohistochemical staining (F, G) showed that TAg protein is present in all cells of the transgenic lens.
View OriginalDownload Slide
Figure 7.
Expression of δenαA-E2F2. A 35S-labeled riboprobe for human E2F2 was used for in situ hybridizations. Bright-field images are shown in (A–F) and the corresponding dark-field images in (A′–F′). A low level of transgene expression was detected in the lens pit of the transgenic mouse at E10.5 (B, B′). By E12.5, transgene expression was detected in both lens epithelial and fiber cells (D, D′). All lens cells continued to express the transgene at E15.5 (F, F′). No signal was detected in the wild-type (WT) lenses. Scale bars, 50 μm.
View OriginalDownload Slide
 
Expression of δenαA-E2F2. A 35S-labeled riboprobe for human E2F2 was used for in situ hybridizations. Bright-field images are shown in (A–F) and the corresponding dark-field images in (A′–F′). A low level of transgene expression was detected in the lens pit of the transgenic mouse at E10.5 (B, B′). By E12.5, transgene expression was detected in both lens epithelial and fiber cells (D, D′). All lens cells continued to express the transgene at E15.5 (F, F′). No signal was detected in the wild-type (WT) lenses. Scale bars, 50 μm.
Figure 7.
 
Expression of δenαA-E2F2. A 35S-labeled riboprobe for human E2F2 was used for in situ hybridizations. Bright-field images are shown in (A–F) and the corresponding dark-field images in (A′–F′). A low level of transgene expression was detected in the lens pit of the transgenic mouse at E10.5 (B, B′). By E12.5, transgene expression was detected in both lens epithelial and fiber cells (D, D′). All lens cells continued to express the transgene at E15.5 (F, F′). No signal was detected in the wild-type (WT) lenses. Scale bars, 50 μm.
Expression of δenαA-E2F2. A 35S-labeled riboprobe for human E2F2 was used for in situ hybridizations. Bright-field images are shown in (A–F) and the corresponding dark-field images in (A′–F′). A low level of transgene expression was detected in the lens pit of the transgenic mouse at E10.5 (B, B′). By E12.5, transgene expression was detected in both lens epithelial and fiber cells (D, D′). All lens cells continued to express the transgene at E15.5 (F, F′). No signal was detected in the wild-type (WT) lenses. Scale bars, 50 μm.
View OriginalDownload Slide
Figure 8.
Expression of δenαA-dnSpry2 transgene. (A) Transgene expression in line LR40. A low level of transgene mRNA was first detected in the lens pit cells at E10.5. At E12.5 transgene expression was found at a high level in both lens epithelial and fiber cells. In situ hybridization signals are shown in both bright-field (left) and dark-field (right) images. (B) Transgene expression in different lines at E13.5. The expression was seen in both lens epithelial and fiber cells, and the levels were higher in lines LR37 and -40 than in line -44. The expression pattern is shown in dark-field images at low magnification (left) and bright-field images at high magnification (right). Scale bars, 50 μm.
View OriginalDownload Slide
 
Expression of δenαA-dnSpry2 transgene. (A) Transgene expression in line LR40. A low level of transgene mRNA was first detected in the lens pit cells at E10.5. At E12.5 transgene expression was found at a high level in both lens epithelial and fiber cells. In situ hybridization signals are shown in both bright-field (left) and dark-field (right) images. (B) Transgene expression in different lines at E13.5. The expression was seen in both lens epithelial and fiber cells, and the levels were higher in lines LR37 and -40 than in line -44. The expression pattern is shown in dark-field images at low magnification (left) and bright-field images at high magnification (right). Scale bars, 50 μm.
Figure 8.
 
Expression of δenαA-dnSpry2 transgene. (A) Transgene expression in line LR40. A low level of transgene mRNA was first detected in the lens pit cells at E10.5. At E12.5 transgene expression was found at a high level in both lens epithelial and fiber cells. In situ hybridization signals are shown in both bright-field (left) and dark-field (right) images. (B) Transgene expression in different lines at E13.5. The expression was seen in both lens epithelial and fiber cells, and the levels were higher in lines LR37 and -40 than in line -44. The expression pattern is shown in dark-field images at low magnification (left) and bright-field images at high magnification (right). Scale bars, 50 μm.
Expression of δenαA-dnSpry2 transgene. (A) Transgene expression in line LR40. A low level of transgene mRNA was first detected in the lens pit cells at E10.5. At E12.5 transgene expression was found at a high level in both lens epithelial and fiber cells. In situ hybridization signals are shown in both bright-field (left) and dark-field (right) images. (B) Transgene expression in different lines at E13.5. The expression was seen in both lens epithelial and fiber cells, and the levels were higher in lines LR37 and -40 than in line -44. The expression pattern is shown in dark-field images at low magnification (left) and bright-field images at high magnification (right). Scale bars, 50 μm.
View OriginalDownload Slide
Copyright 2004 The Association for Research in Vision and Ophthalmology, Inc.
Copyright © 2024 Association for Research in Vision and Ophthalmology.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×
This site uses cookies. By continuing to use our website, you are agreeing to our privacy policy.Accept