Background: 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. Materials and Methods: 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. Results: 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. Conclusions: 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.
Bibliographical noteFunding Information:
This study was supported by IU Health Transplant Institute . The authors thank MRI (Methodist Research Institute) and LARC (Laboratory animal research center) staff for taking care of the animals. This investigation utilized a facility constructed with support from Research Facilities Improvement Program grant number C06RR10601-01 from the National Center for Research Resources, National Institute of Health . All the animals use in this study was approved by Institutional Animal Care and Use Committee (IACUC) .
- fetal liver-derived cells
- genetic engineering