Targeting efficiency of a-1,3-galactosyl transferase gene in pig fetal fibroblast cells

Jin, Dong Il; Lee, Seung-Hyeon; Choi, Jin Hee; Lee, Jae‐Seon; Lee, Jong-Eun; Park, Kwang-Wook; Seo, Jeong‐Sun

doi:10.1038/emm.2003.75

Cited by 22 publications

(16 citation statements)

References 17 publications

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“…(2) Due to the long generation intervals, the derivation of homozygous mutant animals by intercrossing heterozygous animals is a time-consuming process. In previous studies, the low targeting efficiency in primary pig cells has been overcome by gene trapping strategies [25], negative selection [26], or adeno-associated viral vectors [9]. The generation of homozygous knockouts was accelerated by gene conversion where heterozygous mutant cells were cultivated under selective pressure in vitro [27].…”

Section: Discussionmentioning

confidence: 99%

Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis

et al. 2011

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Cystic fibrosis (CF) is the most common lethal inherited disease in Caucasians and is caused by mutations in the CFTR gene. The disease is incurable and medical treatment is limited to the amelioration of symptoms or secondary complications. A comprehensive understanding of the disease mechanisms and the development of novel treatment options require appropriate animal models. Existing CF mouse models fail to reflect important aspects of human CF. We thus generated a CF pig model by inactivating the CFTR gene in primary porcine cells by sequential targeting using modified bacterial artificial chromosome vectors. These cells were then used to generate homozygous CFTR mutant piglets by somatic cell nuclear transfer. The homozygous CFTR mutants lack CFTR protein expression and display severe malformations in the intestine, respiratory tract, pancreas, liver, gallbladder, and male reproductive tract. These phenotypic abnormalities closely resemble both the human CF pathology as well as alterations observed in a recently published CF pig model which was generated by a different gene targeting strategy. Our new CF pig model underlines the value of the CFTR-deficient pig for gaining new insight into the disease mechanisms of CF and for the development and evaluation of new therapeutic strategies. This model will furthermore increase the availability of CF pigs to the scientific community.

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Section: Discussionmentioning

confidence: 99%

Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis

et al. 2011

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“…Unfortunately, to date, authentic pig ES cell lines have not been established. HR in cultured somatic cells is extremely inefficient, and this has become the main barrier in making gene-targeted pigs [3,7,8]. Recently, zinc-finger nuclease (ZFN) technology was shown to be a revolutionary tool for introducing targeted modifications into the genome.…”

mentioning

confidence: 99%

Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer

Bao

Chen

Jong

et al. 2014

Sci. China Life Sci.

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Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs. Conventional gene targeting in pig somatic cells is extremely inefficient. Zinc-finger nuclease (ZFN) technology has been shown to be a powerful tool for efficiently inducing mutations in the genome. However, ZFN-mediated targeting in pigs has rarely been achieved. Here, we used ZFNs to knock out the porcine α-1,3-galactosyl-transferase (GGTA1) gene, which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation. Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1. Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4% and 5.2%, respectively. The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer (SCNT). Three GGTA1 null piglets were born, and one knockout primary fibroblast cell line was established from a cloned fetus. Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane. Functionally, GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum. This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs. GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.pig, xenotransplantation, ZFNs, GGTA1, biallelic knockout, SCNT Citation:

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“…Thus, if the target gene is only active in cell types that are difficult to culture, it is nearly impossible to target the gene locus using this method. Compared with promoter-trapping method, a more widely used strategy includes utilization of both positive and negative selection (PNS) (Jin et al, 2003;Kuroiwa et al, 2004;Richt et al, 2007). A positive selection gene (antibiotic-resistant gene such as neo) in the targeting vector is needed to select cells with an integrated construct a negative selection gene (cytotoxic genes such as the thymidine kinase (TK) gene or the diphtheria toxin A-chain (DT-A) gene) to further select against random integration event.…”

Section: Positive/negative Selection Strategiesmentioning

confidence: 99%

Genetic Modification of Domestic Animals for Agriculture and Biomedical Applications

Yang¹,

Ross²

2012

Biomedical Science, Engineering and Technology

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Targeting efficiency of a-1,3-galactosyl transferase gene in pig fetal fibroblast cells

Cited by 22 publications

References 17 publications

Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis

Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis

Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer

Genetic Modification of Domestic Animals for Agriculture and Biomedical Applications

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