RESUMO
BACKGROUND: The process of selecting transgenic cells has been one of the bottlenecks in the generation of transgenic animals by somatic cell nuclear transfer (SCNT). In particular, selection for the Gal double-knockout (Gal-DKO) genotype has been time consuming and inefficient. The objective of this work was to generate a highly efficient system to select Gal-DKO cells to be used in SCNT without affecting the efficiency in production of Gal-null pigs. MATERIALS AND METHODS: Fetal liver-derived cells deficient in Gal-expression were initially selected by fluorescence-activated cell sorting (FACS) using IB4 conjugated to a fluorescent dye. Cells recovered by FACS were cultured and expanded, followed by a second round of selection using streptavidin magnetic beads and IB4 lectin biotin. RESULTS: Recovery efficiency of target cells was 0.04% for the first selection using FACS and 0.3% for the second round by magnetic beads. Full reprogramming was obtained on selected Gal-DKO cells after FACS and magnetic beads selection, when used for SCNT to produce the Gal-null piglets. Cells obtained from magnetic beads developed 48 colonies; the Gal-null genotype was found in 44 of them (91.7%). Three of these colonies were used to generate piglets by SCNT. From three recipients receiving embryos, two became pregnant and produced 17 piglets, all of them DKO. CONCLUSIONS: Sequential selection of Gal-DKO cells by FACS/magnetic beads is a highly efficient system to generate null cells. Selected cells were successfully used to generate healthy double-knockout piglets by SCNT.
Assuntos
Galactosiltransferases/deficiência , Fígado/citologia , Técnicas de Transferência Nuclear , Animais , Animais Geneticamente Modificados , Células Cultivadas , Citometria de Fluxo , Galactosiltransferases/genética , Fígado/embriologia , Magnetismo , Modelos Animais , SuínosRESUMO
BACKGROUND: Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα-1,3-Gal (Gal) and N-glycolylneuraminic acid (Neu5Gc) are two known xenoantigens. METHODS: We report the homozygous disruption of the α1, 3-galactosyltransferase (GGTA1) and the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes in liver-derived female pig cells using zinc-finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody-binding and antibody-mediated complement-dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal. RESULTS: This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1. CONCLUSIONS: This technology will accelerate the development of pigs for xenotransplantation research.
Assuntos
Dissacarídeos/imunologia , Ácidos Neuramínicos/imunologia , Sus scrofa/genética , Sus scrofa/imunologia , Transplante Heterólogo/imunologia , Animais , Anticorpos Heterófilos/metabolismo , Citotoxicidade Celular Dependente de Anticorpos , Antígenos Heterófilos/imunologia , Antígenos Heterófilos/metabolismo , Sequência de Bases , Células Cultivadas , DNA/genética , Dissacarídeos/deficiência , Feminino , Galactosiltransferases/deficiência , Galactosiltransferases/genética , Técnicas de Inativação de Genes/métodos , Humanos , Leucócitos Mononucleares/imunologia , Oxigenases de Função Mista/deficiência , Oxigenases de Função Mista/genética , Ácidos Neuramínicos/metabolismo , Sus scrofa/metabolismoRESUMO
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.