Skip to main content

Advertisement

SpringerLink
Log in

Screening somatic cell nuclear transfer parameters for generation of transgenic cloned cattle with intragenomic integration of additional gene copies that encode bovine adipocyte-type fatty acid-binding protein (A-FABP)

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Somatic cell nuclear transfer (SCNT) is frequently used to produce transgenic cloned livestock, but it is still associated with low success rates. To our knowledge, we are the first to report successful production of transgenic cattle that overexpress bovine adipocyte-type fatty acid binding proteins (A-FABPs) with the aid of SCNT. Intragenomic integration of additional A-FABP gene copies has been found to be positively correlated with the intramuscular fat content in different farm livestock species. First, we optimized the cloning parameters to produce bovine embryos integrated with A-FABP by SCNT, such as applied voltage field strength and pulse duration for electrofusion, morphology and size of donor cells, and number of donor cells passages. Then, bovine fibroblast cells from Qinchuan cattle were transfected with A-FABP and used as donor cells for SCNT. Hybrids of Simmental and Luxi local cattle were selected as the recipient females for A-FABP transgenic SCNT-derived embryos. The results showed that a field strength of 2.5 kV/cm with two 10-μs duration electrical pulses was ideal for electrofusion, and 4–6th generation circular smooth type donor cells with diameters of 15–25 μm were optimal for producing transgenic bovine embryos by SCNT, and resulted in higher fusion (80%), cleavage (73%), and blastocyst (27%) rates. In addition, we obtained two transgenic cloned calves that expressed additional bovine A-FABP gene copies, as detected by PCR-amplified cDNA sequencing. We proposed a set of optimal protocols to produce transgenic SCNT-derived cattle with intragenomic integration of ectopic A-FABP-inherited exon sequences.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Samiec M, Skrzyszowska M (2011) Transgenic mammalian species, generated by somatic cell cloning, in biomedicine, biopharmaceutical industry and human nutrition/dietetics-recent achievements. Pol J Vet Sci 14(2):317–328

    CAS  PubMed  Google Scholar 

  2. Samiec M, Skrzyszowska M (2011) The possibilities of practical application of transgenic mammalian species generated by somatic cell cloning in pharmacology, veterinary medicine and xenotransplantology. Pol J Vet Sci 14(2):329–340

    CAS  PubMed  Google Scholar 

  3. McCreath KJ et al (2000) Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 405(6790):1066–1069

    Article  CAS  PubMed  Google Scholar 

  4. Schnieke AE et al (1997) Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278(5346):2130–2133

    Article  CAS  PubMed  Google Scholar 

  5. Cibelli JB et al (1998) Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280(5367):1256–1258

    Article  CAS  PubMed  Google Scholar 

  6. Baguisi A et al (1999) Production of goats by somatic cell nuclear transfer. Nat Biotechnol 17(5):456–461

    Article  CAS  PubMed  Google Scholar 

  7. Polejaeva IA et al (2000) Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407(6800):86–90

    Article  CAS  PubMed  Google Scholar 

  8. Lai L et al (2002) Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295(5557):1089–1092

    Article  CAS  PubMed  Google Scholar 

  9. Wilmut I et al (2002) Somatic cell nuclear transfer. Nature 419(6907):583–586

    Article  CAS  PubMed  Google Scholar 

  10. Samiec M, Skrzyszowska M (2012) Roscovitine is a novel agent that can be used for the activation of porcine oocytes reconstructed with adult cutaneous or fetal fibroblast cell nuclei. Theriogenology 78(8):1855–1867

    Article  CAS  PubMed  Google Scholar 

  11. Samiec M, Skrzyszowska M (2014) Biological transcomplementary activation as a novel and effective strategy applied to the generation of porcine somatic cell cloned embryos. Reprod Biol 14(2):128–139

    Article  PubMed  Google Scholar 

  12. Rodriguez-Osorio N et al (2012) Reprogramming mammalian somatic cells. Theriogenology 78(9):1869–1886

    Article  CAS  PubMed  Google Scholar 

  13. Samiec M, Skrzyszowska M, Lipinski D (2012) Pseudophysiological transcomplementary activation of reconstructed oocytes as a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetal fibroblast cells. Pol J Vet Sci 15(3):509–516

    CAS  PubMed  Google Scholar 

  14. Sangalli JR et al (2014) Development to term of cloned cattle derived from donor cells treated with valproic acid. PLoS One 9(6):e101022

    Article  PubMed  PubMed Central  Google Scholar 

  15. Samiec M et al (2015) Trichostatin A-mediated epigenetic transformation of adult bone marrow-derived mesenchymal stem cells biases the in vitro developmental capability, quality, and pluripotency extent of porcine cloned embryos. Biomed Res Int 2015:814686

    Article  PubMed  PubMed Central  Google Scholar 

  16. Samiec M, Skrzyszowska M (2013) Assessment of in vitro developmental capacity of porcine nuclear-transferred embryos reconstituted with cumulus oophorus cells undergoing vital diagnostics for apoptosis detection. Ann Anim Sci 13:513–529

    Google Scholar 

  17. Samiec M, Skrzyszowska M (2013) Creation of cloned pig embryos using contact-inhibited or serum-starved fibroblast cells analysed intra vitam for apoptosis occurrence. Ann Anim Sci 13:275–293

    Google Scholar 

  18. Samiec M, Skrzyszowska M (2013) In vitro development of porcine nuclear-transferred embryos derived from fibroblast cells analysed cytometrically for apoptosis incidence and accuracy of cell cycle synchronization at the G0/G1 stages. Ann Anim Sci 13:735–752

    Google Scholar 

  19. Di Pietro SM, Santome JA (2001) Structural and biochemical characterization of the lungfish (Lepidosiren paradoxa) liver basic fatty acid binding protein. Arch Biochem Biophys 388(1):81–90

    Article  CAS  PubMed  Google Scholar 

  20. Gerbens F et al (1998) The adipocyte fatty acid-binding protein locus: characterization and association with intramuscular fat content in pigs. Mamm Genome 9(12):1022–1026

    Article  CAS  PubMed  Google Scholar 

  21. Jurie C et al (2007) Adipocyte fatty acid-binding protein and mitochondrial enzyme activities in muscles as relevant indicators of marbling in cattle. J Anim Sci 85(10):2660–2669

    Article  CAS  PubMed  Google Scholar 

  22. Li WJ et al (2008) Gene expression of heart- and adipocyte-fatty acid-binding protein and correlation with intramuscular fat in Chinese chickens. Anim Biotechnol 19(3):189–193

    Article  CAS  PubMed  Google Scholar 

  23. Hovenier R et al (1993) Economic values of optimum traits: the example of meat quality in pigs. J Anim Sci 71(6):1429–1433

    CAS  PubMed  Google Scholar 

  24. Gerbens F et al (2001) Associations of heart and adipocyte fatty acid-binding protein gene expression with intramuscular fat content in pigs. J Anim Sci 79(2):347–354

    Article  CAS  PubMed  Google Scholar 

  25. Damon M et al (2006) Number of intramuscular adipocytes and fatty acid binding protein-4 content are significant indicators of intramuscular fat level in crossbred Large White × Duroc pigs. J Anim Sci 84(5):1083–1092

    Article  CAS  PubMed  Google Scholar 

  26. Xu QL ZQ, Chen YL (2011) Cloning, expression and structure modeling analysis of adipocyte fatty acid binding protein gene (FABP4) of sheep. J Agric Biotechnol 19(3):483–489

    Google Scholar 

  27. Li PP GX, Wang JH, Zhang BJ (2009) Progress in the study on correlative candidate gene of fatness traits in pigs. Feed Industry 30(17):47–50

    Google Scholar 

  28. Van Stekelenburg-Hamers AE et al (1993) Nuclear transfer and electrofusion in bovine in vitro-matured/in vitro-fertilized embryos: effect of media and electrical fusion parameters. Mol Reprod Dev 36(3):307–312

    Article  PubMed  Google Scholar 

  29. Zhu J et al (2002) Improvement of an electrical activation protocol for porcine oocytes. Biol Reprod 66(3):635–641

    Article  CAS  PubMed  Google Scholar 

  30. Lai L et al (2006) Generation of cloned transgenic pigs rich in omega-3 fatty acids. Nat Biotechnol 24(4):435–436

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Huang YQ SD, Chen XJ, Li JZ, Zheng SY, Zhan P, Ruan GW, Zhao SH (2011) Influence of morphology, diameter and passage of the donor cells on porcine somatic cell nuclear transfer. Chin Vet Sci 5:530–535

    Google Scholar 

  32. Wang YG ZX, Cheng GX, Cheng Y, Chen JQ, Liu J, Zhang JP, Zhang XC, Lao WD, Zhang SL, Xu SF, Du M (1999) Cloned goats (Capra hircus) from foetal fibroblast cell lines. Chin Sci Bull 45:34–38

    Article  Google Scholar 

  33. Tao T et al (1999) Development of pig embryos by nuclear transfer of cultured fibroblast cells. Cloning 1(1):55–62

    Article  CAS  PubMed  Google Scholar 

  34. Pan DK ZY, Sun XZ, Li Y, Wu CX, Li N (2006) Effects of donor cells on in vitro development of porcine cloned embryos. Acta Veterinaria et Zootechnica Sinica 37(4):331–336

    Google Scholar 

  35. Kubota C et al (1998) In vitro and in vivo survival of frozen-thawed bovine oocytes after IVF, nuclear transfer, and parthenogenetic activation. Mol Reprod Dev 51(3):281–286

    Article  CAS  PubMed  Google Scholar 

  36. Lu FH SD, Wei YM, Pan HP (2005) Studies on the methods of buffalo somatic cells nuclear transfer. Chin J Anim Vet Sci 2:127–132

    Google Scholar 

  37. Kubota C et al (2000) Six cloned calves produced from adult fibroblast cells after long-term culture. Proc Natl Acad Sci USA 97(3):990–995

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Arat S et al (2001) Production of transgenic bovine embryos by transfer of transfected granulosa cells into enucleated oocytes. Mol Reprod Dev 60(1):20–26

    Article  CAS  PubMed  Google Scholar 

  39. Lee GS et al (2003) Improvement of a porcine somatic cell nuclear transfer technique by optimizing donor cell and recipient oocyte preparations. Theriogenology 59(9):1949–1957

    Article  PubMed  Google Scholar 

  40. Hill JR et al (2000) Evidence for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biol Reprod 63(6):1787–1794

    Article  CAS  PubMed  Google Scholar 

  41. Rideout WM 3rd, Eggan K, Jaenisch R (2001) Nuclear cloning and epigenetic reprogramming of the genome. Science 293(5532):1093–1098

    Article  CAS  PubMed  Google Scholar 

  42. Rhind SM et al (2003) Human cloning: can it be made safe? Nat Rev Genet 4(11):855–864

    Article  CAS  PubMed  Google Scholar 

  43. Tian XC (2004) Reprogramming of epigenetic inheritance by somatic cell nuclear transfer. Reprod Biomed Online 8(5):501–508

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the National Science and Technology Major Project “Cultivate new varieties of genetically modified organisms” (Grant No. 2011ZX08008005-003), National Natural Science Foundation of China (Grant No. 31072185), National Natural Science Foundation of China (Grant No. 31272526), the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges Under Beijing Municipality (Grant No. IDHT 20130515) and the Foundation of DA BEI NONG GROUP for Young Teachers (Grant No. 14zk003).

Author information

Author notes

    Authors and Affiliations

    1. College of Animal Science and Technology, Beijing University of Agriculture, Beijing, 102206, China

      Yong Guo, Xihui Sheng, Di Chang, Xiaolong Qi, Xiangguo Wang, Yunhai Liu & Hemin Ni

    2. College of Landscape Design and Forestry, Beijing University of Agriculture, Beijing, 102206, China

      Hejuan Li

    3. Jiahe Hospital of Reproduction Health and Sterility, Qingdao, 266071, China

      Ying Wang

    4. College of Life Science, Northwest University, Xi’an, 710069, China

      Xingrong Yan

    5. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, China

      Junya Li

    Authors
    1. Yong Guo
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Hejuan Li
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Ying Wang
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Xingrong Yan
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Xihui Sheng
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Di Chang
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Xiaolong Qi
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Xiangguo Wang
      View author publications

      You can also search for this author in PubMed Google Scholar

    9. Yunhai Liu
      View author publications

      You can also search for this author in PubMed Google Scholar

    10. Junya Li
      View author publications

      You can also search for this author in PubMed Google Scholar

    11. Hemin Ni
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Hemin Ni.

    Additional information

    Yong Guo and Hejuan Li have contributed equally to this work.

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Guo, Y., Li, H., Wang, Y. et al. Screening somatic cell nuclear transfer parameters for generation of transgenic cloned cattle with intragenomic integration of additional gene copies that encode bovine adipocyte-type fatty acid-binding protein (A-FABP). Mol Biol Rep 44, 159–168 (2017). https://doi.org/10.1007/s11033-016-4094-8

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s11033-016-4094-8

    Keywords

    Search

    Navigation