A Weakened Transcriptional Enhancer Yields Variegated Gene Expression
Figure 1
Structure of the reporter cassettes
a) The endogenous μ gene.
The boxes labeled V and C represent the exons encoding the variable (V) and constant (Cμ) regions of the immunoglobulin μ heavy chain gene.
The relative positions of the intronic enhancer (Eμ) and switch (Sμ) regions in the V-C intron are shown.
The Eμ enhancer is depicted with three components: the core enhancer (E) flanked by matrix attachment regions, M and M′.
b) Recombination-mediated cassette exchange.
The upper panel depicts a DNA segment in which (inverted) LoxP sites (1L and L1) flank a gene encoding the HyTK fusion protein (hygromycin-resistance and thymidine kinase [gancyclovir sensitivity]).
As described previously, this DNA segment was inserted in the genome of the recipient hybridoma cell line [9].
The HyTK and μ cassettes are represented as thick lines, with major exons as rectangles, the LoxP sites as triangles (L1 in the “forward” orientation, 1L in the “reverse” orientation).
The three-stranded line represents the chromosomal DNA.
The residual backbone of the vector that is shared between the target and the replacement is represented as a thin line; the remainder of the reporter cassette is represented as a dotted line.
The middle panel depicts the structure of the replacement vector designed to substitute a modified μ gene for the Hy-TK gene via Cre-mediated recombination of the LoxP sites.
This vector lacks the switch (Sμ) region and was constructed by joining two segments of the μ gene of the Sp6 hybridoma: The 2.0 kb V-bearing segment includes the DNA between the PacI site (∼850 bp 5′ of the initiator ATG) and the NgoMIV site 3′ of J4.
The 4.6 kb Cμ-bearing segment includes the DNA between the SnaI site 5′ of Cμ and the SphI site 3′ of Cμ.
DNA segments were inserted either in the intron at the NgoMIV site (denoted i), 1.2 kb 3′ of the initiator ATG, or 3′ of Cμ at the SphI site (denoted 3′), 5.9 kb 3′ of the ATG.
The lower panel depicts the structure after the μ reporter cassette has replaced the HyTK gene.
To distinguish replacements from random insertions we made use of the HinDIII (H) and NheI (N) sites that distinguish the DNA that flanks the HyTK and μ genes.
The notations (i) and (3′) indicate the two sites where enhancer-derived segments were inserted.
c) Structure of the reporter gene used for assaying insulator segments.
d) The enhancer-derived segments.
The “full” enhancer corresponds to the 2034 bp DNA segment bounded by the NgoMIV and Bst1107I sites, which are denoted as nucleotides 1 and 2034, respectively.
The indicated subsegments were prepared by PCR, and nucleotide positions of their endpoints, numbered from the first nucleotide of the NgoMIV site are as follows: M, 1-782; E, 783-1035; M′, 1036-2034; p, 604-782; p′, 1036-1295; q′, 1296-1342; r′, 1343-1654; s′, 1655-1976.
The XbaI sites that are often used to delimit the MARs are at 448 and 1441.
The Bright binding sites are P1, 624-648; P2, 733-767; P4 1183-1202; P4, 1237-1276.
e) Subsegments of the gpt cassette.
The full gpt expression cassette includes the SV40 promoter (S), the gpt structural gene (gpt) and the SV40 polyA site (T).
The gpt structural gene was divided into three subsegments, denoted x, y z.
The nucleotide positions are measured from the first nucleotide of the SphI site in the SV40 promoter.
The figures are not to scale.