Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
J Surg Res. 2011 Dec;171(2):e223-9. doi: 10.1016/j.jss.2011.07.051. Epub 2011 Aug 25.
Since there are no pig embryonic stem cells, pig genetic engineering is done in fetal fibroblasts that remain totipotent for only 3 to 5 wk. Nuclear donor cells that remain totipotent for longer periods of time would facilitate complicated genetic engineering in pigs. The goal of this study was to test the feasibility of using fetal liver-derived cells (FLDC) to perform gene targeting, and create a genetic knockout pig.
FLDC were isolated and processed using a human liver stem cell protocol. Single copy α-1,3-galactosyl transferase knockout (GTKO) FLDCs were created using electroporation and neomycin resistant colonies were screened using PCR. Homozygous GTKO cells were created through loss of heterozygosity mutations in single GTKO FLDCs. Double GTKO FLDCs were used in somatic cell nuclear transfer (SCNT) to create GTKO pigs.
FLDCs grew for more than 80 population doublings, maintaining normal karyotype. Gene targeting and loss of heterozygosity mutations produced homozygous GTKO FLDCs. FLDCs used in SCNT gave rise to homozygous GTKO pigs.
FDLCs can be used in gene targeting and SCNT to produce genetically modified pigs. The increased life span in culture compared to fetal fibroblasts may facilitate genetic engineering in the pig.
由于没有猪胚胎干细胞，猪的基因工程是在胎儿成纤维细胞中进行的，这些细胞仅保持全能性 3 到 5 周。能够保持更长时间全能性的核供体细胞将有助于在猪中进行复杂的基因工程。本研究的目的是测试使用胎肝来源的细胞（FLDC）进行基因靶向并创建基因敲除猪的可行性。
使用人类肝干细胞方案分离和处理 FLDC。使用电穿孔和新霉素抗性菌落筛选创建单拷贝α-1,3-半乳糖基转移酶敲除（GTKO）FLDC。通过单 GTKO FLDC 中的杂合丢失突变产生纯合 GTKO 细胞。使用双 GTKO FLDC 进行体细胞核移植（SCNT）以创建 GTKO 猪。
FLDC 生长超过 80 个倍增，保持正常核型。基因靶向和杂合丢失突变产生了纯合 GTKO FLDC。用于 SCNT 的 FLDC 产生了纯合 GTKO 猪。
FDLC 可用于基因靶向和 SCNT 以生产基因修饰猪。与胎儿成纤维细胞相比，培养物中的寿命延长可能有助于猪的基因工程。
