Deletion of the α(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep

Denning, Chris; Burl, Sarah; Ainslie, A.; Bracken, John; Dinnyés, András; Fletcher, Judy; King, Tim; Ritchie, M.; Ritchie, William A.; Rollo, M; Sousa, Paul A. De; Travers, A; Wilmut, Ian; Clark, Anthony

doi:10.1038/89313

Cited by 252 publications

(120 citation statements)

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“…Overall, frequency of homologous recombination at each targeting step was sufficiently high (0.4-6.4%) to produce at least a couple of targeted colonies from ∼500 selected colonies that were screened by PCR in each experiment. The efficiency might be attributed to several conditions that were optimized specifically for bovine fibroblast targeting, including using appropriate promoters to maximize expression of positive selection marker genes, using the DT-A gene for negative selection 19 , using contiguous regions of homology in the targeted gene loci, optimizing electroporation conditions 5 and cloning immediately after PCR selection with a modified system to facilitate reprogramming of the donor cells 20 .…”

Section: Discussionmentioning

confidence: 99%

“…Gene targeting is accomplished in the mouse using embryonic stem (ES) cells, but in essentially all other species, ES cells suitable for gene targeting are not available. The few reports on gene targeting in other mammalian species used primary somatic cells followed by embryonic cloning [4][5][6][7] ; in some instances, the embryos were then used to produce cloned offspring. Gene targeting in primary somatic cells is a challenge [8][9][10][11][12] because somatic cells have a relatively short lifespan, which limits selection of properly targeted cell colonies, and a low frequency of homologous recombination 11 compared with mouse ES cells.…”

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confidence: 99%

“…Because of these limitations, success in somatic cell gene targeting has been achieved for only a couple of genes that were transcriptionally active in the cell line used for targeting and only in sheep and pig. Transcriptionally active genes are more amenable to gene targeting than silent genes, because they have a higher frequency of homologous recombination 5,8 and correctly targeted cells can be easily selected by having the targeted gene promoter drive expression of a selection marker. Application of this 'promoter-less' positive selection 4-7 is limited to transcriptionally active genes in the somatic cells.…”

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Sequential targeting of the genes encoding immunoglobulin-μ and prion protein in cattle

et al. 2004

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Gene targeting is accomplished using embryonic stem cells in the mouse but has been successful, only using primary somatic cells followed by embryonic cloning, in other species. Gene targeting in somatic cells versus embryonic stem cells is a challenge; consequently, there are few reported successes and none include the targeting of transcriptionally silent genes or double targeting to produce homozygotes. Here, we report a sequential gene targeting system for primary fibroblast cells that we used to knock out both alleles of a silent gene, the bovine gene encoding immunoglobulin-µ (IGHM), and produce both heterozygous and homozygous knockout calves. We also carried out sequential knockout targeting of both alleles of a gene that is active in fibroblasts, encoding the bovine prion protein (PRNP), in the same genetic line to produce doubly homozygous knockout fetuses. The sequential gene targeting system we used alleviates the need for germline transmission for complex genetic modifications and should be broadly applicable to gene functional analysis and to biomedical and agricultural applications.

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Section: Discussionmentioning

confidence: 99%

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Sequential targeting of the genes encoding immunoglobulin-μ and prion protein in cattle

et al. 2004

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“…Nuclear transfer of somatic cells in pigs is the most promising procedure to achieve knockout of α-1,3-galactosyl transferase (13-GT) gene for pigto-human xenotransplatation (Betthanser et al, 2000;Onishi, et al, 2000;Polejaeva, et al, 2000;Harrison et al, 2002). Production of viable pig and sheep with targeted insertion at several gene loci following nuclear transfer with gene-targeted cells has been reported (McCreath et al, 2000;Denning et al, 2001;Dai et al, 2002;Lai et al, 2002;Phelps et al, 2003;Ramsoondar et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

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

Jin

Lee²,

Choi³

et al. 2003

Exp Mol Med

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“…Unfortunately, because of its high degree of specificity, the frequency of homologous recombination is very low and varies from 10 -7 to 10 -8 in primary somatic cells (39 -41). Although it has not been possible to target some loci because of this inefficiency (42), there have been reports of successful targeting in somatic cells and production of genetically modified animals by using the same strategy as used in mouse embryonic stem cells (43)(44)(45). Recently chimeric nucleases have been used to stimulate gene targeting in human somatic cells by creating double strandedbreaks in the genomic target (46).…”

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Somatic Cell Cloning

Piedrahita¹,

Mir²,

Dindot³

et al. 2004

Journal of the American Society of Nephrology

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Abstract. With the increasing difficulties associated with meeting the required needs for organs used in transplantation, alternative approaches need to be considered. These include the use of stem cells as potential sources of specialized cells, the ability to transdifferentiate cell types in culture, and the development of complete organs that can be used in humans. All of the above goals will require a complete understanding of the factors affecting cell differentiation and nuclear reprogramming. To make this a reality, however, techniques associated with cloning and genetic modifications in somatic cells need to be continued to be developed and optimized. This includes not only an enhancement of the rate of homologous recombination in somatic cells, but also a thorough understanding of the nuclear reprogramming process taking place during nuclear transfer. The understanding of this process is likely to have an effect beyond the area of nuclear transfer and assist with better methods for transdifferentiation of mammalian cells.The ever-expanding gap between availability of organs and the number of patients awaiting an organ transplant is of great concern. In 2003, 14,880 transplantations were carried out between January and August, yet more than 83,000 patients were in the organ waiting list (based on data from the Organ Procurement and Transplantation Network as of November 1, 2003). This increasing divergence between availability of organ donors and the increasing number of patients in waiting lists has caused researchers to look into alternative methods to fulfill these needs. Whereas for complete organ replacement, xenotransplantation with pig organs is the main alternative, for tissue repair, other alternatives exist. The most promising to date is the use of embryo-derived and adult-derived stem cells (1). Cloning by nuclear transfer has a key role to play both by assisting in the generation of swine with complex genetic manipulations, and in the generation of human stem cells. Although ethical concerns need to be considered for the application of these technologies, such discussion is beyond the scope of this review. This review will focus on the development of the cloning technology, the basic concept in nuclear reprogramming, and recent advances in both understanding the epigenetic process of nuclear reprogramming and in enhancing the ability to undertake complex genetic manipulation in this species.The goals of generating a complete organism by nuclear transfer of a nuclei into an oocyte, or reproductive cloning, has been pursued for many years. By use of frog (Rana pipiens) eggs, Briggs and King (2) described the first nuclear transfer experiment. Briefly, ovulated MII eggs were enucleated with a handmade needle, and cells from a developing frog were lysed and their nuclei injected into the egg. Over the next 4 to 11 d, the majority of the cleaved blastulae developed into normal postneurula and tadpole stages, demonstrating the ability of the injected nuclei to at least partially complete normal de...

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Deletion of the α(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep

Cited by 252 publications

References 22 publications

Sequential targeting of the genes encoding immunoglobulin-μ and prion protein in cattle

Sequential targeting of the genes encoding immunoglobulin-μ and prion protein in cattle

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

Somatic Cell Cloning

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