Non-viral transfection of goat germline stem cells by nucleofection results in production of transgenic sperm after germ cell transplantation
| Autor: | Margaret M. Destrempes, Susan Megee, Lin Tang, William G. Gavin, Mark Modelski, David Melican, Ina Dobrinski, Susan A. Overton, Jinping Luo, Yann Echelard, S. Blash, Wenxian Zeng, Alla Bondareva, Sandra L. Ayres |
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| Rok vydání: | 2012 |
| Předmět: |
Male
endocrine system Genotype Transgene Nucleofection beta-Globins Biology Transfection Article Animals Genetically Modified Testis Genetics medicine Animals Humans Transgenes Promoter Regions Genetic Human Growth Hormone Goats Stem Cells fungi Caseins Cell Biology Immunohistochemistry Spermatozoa Molecular biology Sperm Transgenesis Transplantation Germ Cells medicine.anatomical_structure Female Stem cell Chickens Germ cell Stem Cell Transplantation Developmental Biology |
| Zdroj: | Molecular Reproduction and Development. 79:255-261 |
| ISSN: | 1040-452X |
| Popis: | Germline stem cells (GSCs) can be used for large animal transgenesis, in which GSCs that are genetically manipulated in vitro are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objectives of this study were to explore a non-viral approach for transgene delivery into goat GSCs and to investigate the efficiency of nucleofection in producing transgenic sperm. Four recipient goats received fractionated irradiation at 8 weeks of age to deplete endogenous GSCs. Germ cell transplantations were performed 8–9 weeks post-irradiation. Donor cells were collected from testes of 9-week-old goats, enriched for GSCs by Staput velocity sedimentation, and transfected by nucleofection with a transgene construct harboring the human growth hormone gene under the control of the goat beta-casein promoter (GBC) and a chicken beta-globin insulator (CBGI) sequence upstream of the promoter. For each recipient, transfected cells from 10 nucleofection reactions were pooled, mixed with non-transfected cells to a total of 1.5 × 108 cells in 3 ml, and transplanted into one testis (n = 4 recipients) by ultrasound-guided cannulation of the rete testis. The second testis of each recipient was removed. Semen was collected, starting at 9 months after transplantation, for a period of over a year (a total of 62 ejaculates from four recipients). Nested genomic PCR for hGH and CBGI sequences demonstrated that 31.3% ± 12.6% of ejaculates were positive for both hGH and CBGI. This study provides proof-of-concept that non-viral transfection (nucleofection) of primary goat germ cells followed by germ cell transplantation results in transgene transmission to sperm in recipient goats. Mol. Reprod. Dev. 79: 255–261, 2012. © 2011 Wiley Periodicals, Inc. |
| Databáze: | OpenAIRE |
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