Kim K-J, Cho C M, Kim B-G, Lee Y-A, Kim B-J, Kim Y-H, Kim C G, Schmidt J A, Ryu B-Y
Department of Animal Science and Technology, Chung-Ang University, Ansung, Gyeonggi-do 456-756, Korea.
J Anim Sci. 2014 Jan;92(1):106-18. doi: 10.2527/jas.2013-6885. Epub 2013 Oct 28.
Undifferentiated germ cells have the capacity to develop into sperm capable of fertilizing oocytes and contributing genetic material to subsequent generations. The most primitive prepubertal undifferentiated germ cells include gonocytes and undifferentiated spermatogonia, including spermatogonial stem cells (SSC). Gonocytes, present in the testis at birth, differentiate into SSC, which maintain spermatogenesis for the remainder of the male's life. Because of their capacity to contribute to lifelong spermatogenesis, undifferentiated germ cells are attractive targets for genetic modification to produce transgenic animals, including cattle. To maximize the efficiency of genetic modification of bovine gonocytes and SSC, effective enrichment techniques need to be developed. Selection of bovine gonocytes using differential plating was improved 8-fold (P < 0.001) when using a combination of extracellular matrix proteins, including laminin, fibronectin, collagen type IV, and gelatin, compared to using laminin and gelatin alone. Selected cells labeled with PKH26 formed colonies of donor-derived germ cells after transplantation into recipient mouse testes, indicating putative stem cell function. Significantly more colonies (P < 0.001) per 1 × 10(5) viable transplanted cells were formed from isolated nonadherent cells (203 ± 23.2) compared to adherent (20 ± 2.7) or Percoll (45.5 ± 4.5) selected cells. After selection, some gonocytes were transduced using a lentiviral vector containing the transgene for the enhanced green fluorescent protein. Transduction efficiency was 17%. Collectively, these data demonstrate effective methods for the selection and genetic modification of bovine undifferentiated germ cells.
未分化的生殖细胞有能力发育成能够使卵母细胞受精并为后代贡献遗传物质的精子。最原始的青春期前未分化生殖细胞包括生殖母细胞和未分化的精原细胞，其中包括精原干细胞（SSC）。出生时存在于睾丸中的生殖母细胞会分化为精原干细胞，后者在雄性的余生中维持精子发生。由于其对终身精子发生的贡献能力，未分化的生殖细胞是进行基因改造以生产转基因动物（包括牛）的有吸引力的靶标。为了最大限度地提高牛生殖母细胞和精原干细胞的基因改造效率，需要开发有效的富集技术。与单独使用层粘连蛋白和明胶相比，使用包括层粘连蛋白、纤连蛋白、IV型胶原和明胶在内的细胞外基质蛋白组合时，利用差异铺板法选择牛生殖母细胞的效率提高了8倍（P < 0.001）。用PKH26标记的选定细胞在移植到受体小鼠睾丸后形成了供体来源的生殖细胞集落，表明具有假定的干细胞功能。与贴壁（20 ± 2.7）或Percoll（45.5 ± 4.5）选择的细胞相比，每1×10(5)个活的移植细胞中，从分离的非贴壁细胞（203 ± 23.2）形成的集落明显更多（P < 0.001）。选择后，一些生殖母细胞使用含有增强型绿色荧光蛋白转基因的慢病毒载体进行转导。转导效率为17%。总体而言，这些数据证明了牛未分化生殖细胞选择和基因改造的有效方法。
