title: Gene targeting and cloning in pigs using fetal liver derived cells.
abstract: 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.
  
  
  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.
authors.affiliation: Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
authors.forename: Sanjeev K
authors.initials: SK
authors.lastname: Waghmare
authors.forename: Jose
authors.initials: J
authors.lastname: Estrada
authors.forename: Luz
authors.initials: L
authors.lastname: Reyes
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Bess
authors.initials: B
authors.lastname: Ivary
authors.forename: Richard A
authors.initials: RA
authors.lastname: Sidner
authors.forename: Chris
authors.initials: C
authors.lastname: Burlak
authors.forename: A Joseph
authors.initials: AJ
authors.lastname: Tector
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D018929:Cell Culture Techniques / Q000379:methods; D019976:Cloning, Organism / Q000379:methods*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology; D005240:Feasibility Studies; D005260:Female; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D055786:Gene Knockout Techniques; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods; D006720:Homozygote; D008099:Liver / Q000166:cytology*; D019656:Loss of Heterozygosity / Q000235:genetics; D008297:Male; D053652:Nuclear Transfer Techniques; D011247:Pregnancy; D013552:Swine / Q000235:genetics*; D013623:Tail / Q000166:cytology; D014183:Transplantation, Heterologous