Indian Journal of Science and Technology

Open Access Open Access  Restricted Access Subscription Access

Production of Transgenic Bovine Embryos by Microinjection Method of a Lentiviral Vector in Zygotes


Affiliations
1 University of Sucre, Sincelejo, Colombia
 

Objective: To produce bovine transgenic embryos by microinjection of a lentiviral vector that carries the eGFP gene as a marker in zygotes six hours after fertilization. Methods: 834 oocytes were matured and subjected to one of four treatments designed as follows: CC: Control: IVF with Cumulus-oocyte with (COCs), cultivated in CR2 medium supplemented with 10% FBS and incubated at 38.5°C in atmosphere of 95% humidity and 5% CO₂.CCM: Control culture medium: fertilized in vitro for six hours, cultured in medium SOF aluminum in pouches under the same conditions of CC. MC: Microinjection control: Fertilized under the same treatment conditions CCM. After six hours they were microinjected with TALP medium and cultured in sachets with the same conditions of CCM treatment. ML: Microinjected with the lentivirus: Fertilized in the same conditions of the CCM treatment. After six hours they were microinjected with the lentiviral vector carrying the eGFP transgene and cultured in sachets with the same treatment conditions CCM and MC. Findings: The cleavage rate found in CC was higher (p < 0.05) than that observed in the other treatments. The rates of blastocysts found between CC, CCM and MC did not differ significantly (p > 0.05) in them, but yes, with ML (p < 0.05). On average, 76.4% of the zygotes obtained in ML expressed the green fluorescent protein. Application/Improvements: The cult ure conditions used were suitable for CC, CCM and MC, microinjection with lentiviral vector has some influence on embryo development, it succeeded in obtaining transgenic zygotes.
User

  • Laible G. Production of transgenic livestock: Overview of transgenic technologies. Animal Biotechnology; 2018. p. 95–121.

  • Otero R, Hernandez D, Camargo LS de A. Production of bovine transgenic embryos by microinjection of a lentiviral vector in mature ovocytes. Indian Journal of Science and Technology. 2018; 11(31):1–8. https://doi.org/10.17485/ijst/2018/v11i31/130839

  • FELASA Working Group, Rulicke T, Montagutelli X, Pintado B, Thon R, Hedrich HJ. FELASA guidelines for the production and nomenclature of transgenic rodents. Laboratory Animals. 2007; 41(3):301–11. PMid: 17640457. https://doi.org/10.1258/002367707781282758

  • Felmer R. Animales transgenicos: Pasado, presente y futuro. Archivos de Medicina Veterinaria. 2004; 36(2):105–17. https://doi.org/10.4067/S0301732X2004000200002

  • Monzani PS, Adona PR, Ohashi OM, Meirelles FV, Wheeler MB. Transgenic bovine as bioreactors: Challenges and perspectives. Bioengineered. 2016; 7(3):123–31. PMid: 27166649 PMCid: PMC4927206. https://doi.org/10.1080/21655979.2016.1171429

  • Rumpf R, Melo E. Producao de animais transgênicos: Metodologia e aplicacoes. Brasil: Embrapa Recurso Genetico e Biotecnologia; 2005. p. 1–27.

  • Sosa MAG, Gasperi RD, Elder GA. Animal transgenesis: An overview. Brain Structure and Function. 2010; 214(2– 3):91–109. PMid: 19937345. https://doi.org/10.1007/s00429-009-0230-8

  • Houdebine LM. Transgenesis to improve animal production. Livestock Production Science. 2002; 74(3):255–68. https://doi.org/10.1016/S0301-6226(02)00018-0

  • Menoret S, Tesson L, Remy S, Usal C, Ouisse L, Brusselle L. Transgenic animals and genetic engineering techniques. Transgenic Research. 2015; 24(6):1079–85. PMid: 26358113. https://doi.org/10.1007/s11248-015-9904-6

  • Kues WA, Niemann H. Advances in farm animal transgenesis. Preventive Veterinary Medicine. 2011; 102(2):146–56. PMid: 21601297. https://doi.org/10.1016/j.prevetmed.2011.04.009

  • Chu VT, Graf R, Wirtz T, Weber T, Favret J, Li X. Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line. PNAS. 2016; 113(44):12514–9. PMid: 27729526 PMCid: PMC5098665. https://doi.org/10.1073/ pnas.1613884113

  • Yum SY, Lee SJ, Park SG, Shin IG, Hahn SE, Choi WJ. Long-term health and germline transmission in transgenic cattle following transposon-mediated gene transfer. BMC Genomics. 2018; 19(1):387. PMid:29792157 PMCid:PMC5966871. https://doi.org/10.1186/s12864-018-4760-4

  • Curcio AG, Bressan FF, Meirelles FV, Dias AJB. Achievements and perspectives in cloned and transgenic cattle production by nuclear transfer: influence of cell type, epigenetic status and new technology. Animal Reproduction. 2017; 14(4):1003–13. https://doi.org/10.21451/1984-3143-AR853

  • Wang Y, Song Y, Liu Q, Liu C, Wang L, Liu Y. Quantitative analysis of lentiviral transgene expression in mice over seven generations. Transgenic Research. 2010; 19(5):775–84. PMid: 20091347. https://doi.org/10.1007/ s11248-009-9355-z

  • Crispo M, Vilari-o M, Santos-Neto PC dos, Nu-ezOlivera R, Cuadro F, Barrera N. Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis. Transgenic Research. 2015; 24(1):31–41. PMid: 25048992. https://doi.org/10.1007/s11248-014-9816-x

  • Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992; 111(2):229–33. https://doi.org/10.1016/0378-1119(92)90691-H

  • Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science. 2002; 295(5556):868–72. PMid: 11786607. https://doi.org/10.1126/science.1067081

  • Hofmann A, Zakhartchenko V, Weppert M, Sebald H, Wenigerkind H, Brem G. Generation of transgenic cattle by lentiviral gene transfer into oocytes. Biology of Reproduction. 2004; 71(2):405–9. PMid: 15044266. https:// doi.org/10.1095/biolreprod.104.028472

  • Miao K, Guo M, An L, Xu XL, Wu H, Wang D. A new method to efficiently produce transgenic embryos and mice from low-titer lentiviral vectors. Transgenic Research. 2011; 20(2):357–63. PMid: 20585977. https://doi.org/10.1007/ s11248-010-9414-5

  • Van Soom A, Wrathall AE, Herrler A, Nauwynck HJ. Is the zona pellucida an efficient barrier to viral infection? Reproduction, Fertility and Development. 2010; 22(1):21– 31. PMid: 20003842. https://doi.org/10.1071/RD09230

  • Samaniego J, Ayala L, Nieto P, Rodas R, Vazquez J, Murillo Y. Competencia del ovocito bovino obtenido por Ovum pickup valorado mediante el azul brillante de Cresilo. Revista de Investigaciones Veterinarias del Peru. 2018; 29(2):77–80.

  • Otero R, Hernandez D, Camargo LS de A. Effect of Trichostatin-A on embryons of bovine clones modified genetically with GFP. Indian Journal of Science and Technology. 2018; 11(25):1–9. https://doi.org/10.17485/ijst/2018/v11i25/128251

  • Pfeifer A, Ikawa M, Dayn Y, Verma IM. Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proceedings of the National Academy of Sciences USA. 2002; 99(4):2140–5. PMid: 11854510 PMCid: PMC122332 https://doi.org/10.1073/pnas.251682798

  • Hofmann A, Kessler B, Ewerling S, Weppert M, Vogg B, Ludwig H. Efficient transgenesis in farm animals by lentiviral vectors. EMBO Reports. 2003; 4(11):1054–60. PMid: 14566324 PMCid: PMC1326377. https://doi.org/10.1038/sj.embor.7400007

  • Whitelaw CBA, Radcliffe PA, Ritchie WA, Carlisle A, Ellard FM, Pena RN. Efficient generation of transgenic pigs using Equine Infectious Anaemia Virus (EIAV) derived vector. FEBS Letters. 2004; 571(1–3):233–6. PMid: 15280048. https://doi.org/10.1016/j.febslet. 2004.06.076

  • Chan AW, Chong KY, Martinovich C, Simerly C, Schatten G. Transgenic monkeys produced by retroviral gene transfer into mature oocytes. Science. 2001; 291(5502):309–12. PMid: 11209082. https://doi.org/10.1126/science. 291.5502.309

  • Pfeifer A. Lentiviral transgenesis. Transgenic Research. 2004; 13(6):513–22. PMid: 15672832. https://doi.org/10.1007/s11248-004-2735-5

  • Craigie R. Nucleoprotein Intermediates in HIV-1 DNA Integration: Structure and function of HIV-1 Intasomes. Virus Protein and Nucleoprotein Complexes. 2018; 88:189–210. PMid: 29900498. https://doi.org/10.1007/978981-10-8456-0_9

  • Gonçalves J, Moreira E, Sequeira IJ, Rodrigues AS, Rueff J, Bras A. Integration of HIV in the human genome: Which sites are preferential? A Genetic and Statistical Assessment. International Journal of Genomics; 2016. p. 1–6. PMid: 27294106 PMCid: PMC4880676. https://doi.org/10.1155/2016/2168590

  • Xu YN, Uhm SJ, Koo BC, Kwon MS, Roh JY, Yang JS. Production of transgenic Korean native cattle expressing enhanced green fluorescent protein using a FIV-based lentiviral vector injected into MII oocytes. Journal of Genetics and Genomics. 2013; 40(1):37–43. PMid: 23357343 https://doi.org/10.1016/j.jgg.2012.11.001

  • Wu L, Zhang C, Zhang J. HMBOX1 negatively regulates NK cell functions by suppressing the NKG2D/DAP10 signaling pathway. Cellular and Molecular Immunology. 2011 Sep; 8(5):433–40. PMid: 21706044 PMCid: PMC4012885. https://doi.org/10.1038/cmi.2011.20

  • Zhang Z, Sun P, Yu F, Yan L, Yuan F, Zhang W. Transgenic quail production by microinjection of lentiviral vector into the early embryo blood vessels. PLoS ONE. 2012; 7(12):e50817. PMid: 23251391 PMCid: PMC3520935. https://doi.org/10.1371/journal.pone.0050817

  • Tian Y, Li W, Wang L, Liu C, Lin J, Zhang X. Expression of 2A peptide mediated tri-fluorescent protein genes were regulated by epigenetics in transgenic sheep. Biochemical and Biophysical Research Communications. 2013; 434(3):681–7. PMid: 23603255. https://doi.org/10.1016/j.bbrc.2013.04.009

  • Singer O, Verma IM. Applications of lentiviral vectors for shRNA delivery and transgenesis. Current Gene Therapy. 2008; 8(6):483–8. PMid: 19075631 PMCid: PMC2774780. https://doi.org/10.2174/156652308786848067

  • Reichenbach M, Lim T, Reichenbach H-D, Guengoer T, Habermann FA, Matthiesen M. Germ-line transmission of lentiviral PGK-EGFP integrants in transgenic cattle: New perspectives for experimental embryology. Transgenic Research. 2010; 19(4):549–56. PMid: 19862638. https://doi.org/10.1007/s11248-009-9333-5

  • Park F. Lentiviral vectors: Are they the future of animal transgenesis? Physiological Genomics. 2007; 31(2):159–73. PMid: 17684037. https://doi.org/10.1152/physiolgenomics.00069.2007

  • Pfeifer A, Lim T, Zimmermann K. Chapter one - Lentivirus transgenesis. methods in enzymology. 2010; 477:3–15. https://doi.org/10.1016/S0076-6879(10)77001-4


Abstract Views: 216

PDF Views: 0




  • Production of Transgenic Bovine Embryos by Microinjection Method of a Lentiviral Vector in Zygotes

Abstract Views: 216  |  PDF Views: 0

Authors

Rafael Otero
University of Sucre, Sincelejo, Colombia
Darwin Hernandez
University of Sucre, Sincelejo, Colombia
Donicer Montes
University of Sucre, Sincelejo, Colombia

Abstract


Objective: To produce bovine transgenic embryos by microinjection of a lentiviral vector that carries the eGFP gene as a marker in zygotes six hours after fertilization. Methods: 834 oocytes were matured and subjected to one of four treatments designed as follows: CC: Control: IVF with Cumulus-oocyte with (COCs), cultivated in CR2 medium supplemented with 10% FBS and incubated at 38.5°C in atmosphere of 95% humidity and 5% CO₂.CCM: Control culture medium: fertilized in vitro for six hours, cultured in medium SOF aluminum in pouches under the same conditions of CC. MC: Microinjection control: Fertilized under the same treatment conditions CCM. After six hours they were microinjected with TALP medium and cultured in sachets with the same conditions of CCM treatment. ML: Microinjected with the lentivirus: Fertilized in the same conditions of the CCM treatment. After six hours they were microinjected with the lentiviral vector carrying the eGFP transgene and cultured in sachets with the same treatment conditions CCM and MC. Findings: The cleavage rate found in CC was higher (p < 0.05) than that observed in the other treatments. The rates of blastocysts found between CC, CCM and MC did not differ significantly (p > 0.05) in them, but yes, with ML (p < 0.05). On average, 76.4% of the zygotes obtained in ML expressed the green fluorescent protein. Application/Improvements: The cult ure conditions used were suitable for CC, CCM and MC, microinjection with lentiviral vector has some influence on embryo development, it succeeded in obtaining transgenic zygotes.

References





DOI: https://doi.org/10.17485/ijst%2F2018%2Fv11i41%2F131659