Gene Targeting in Primary Fetal Fibroblasts from Sheep and Pig

Denning, Chris; Dickinson, Paul; Burl, Sarah; Wylie, Diana; Fletcher, Judy; Clark, Anthony

doi:10.1089/15362300152725945

Cited by 64 publications

(48 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nuclear transfer with cultured somatic cells was developed as an alternative [1], but is technically much more difficult. More than 10 years after our first demonstration, there are still few examples of gene targeting in mammals other than mice: COL1A1 in sheep [1], PRNP in sheep, cattle and goats [2][3][4], GGTA1 in pigs [5,6], IGH in cattle [3] and CFTR in pigs [7]. Posttranscriptional gene silencing or 'gene knockdown' by RNA interference (RNAi) can mimic loss-of-function mutations or gene knockout in mice [8].…”

Section: Introductionmentioning

confidence: 99%

RNA Interference in Pigs: Comparison of RNAi Test Systems and Expression Vectors

et al. 2010

View full text Add to dashboard Cite

We have examined the use of RNA interference as a means of downregulating gene expression and provide the first comparison of shRNA and artificial miRNA constructs for transgenic livestock. Several in vitro assays were performed to identify the most effective RNAi constructs. shRNA and miRNA constructs achieved significant downregulation of two porcine target genes: the milk whey protein beta-lactoglobulin and the tumour suppressor p53. Results of different assays were, however, sometimes at variance, indicating that no one assay can be relied upon to predict the effectiveness of an RNAi construct. Our findings are that screening of RNAi constructs is most informative if carried out in primary cells that express the target gene and are competent for somatic cell nuclear transfer. Importantly, the use of miRNA constructs makes tissue specific gene knockdown in large animals a realistic possibility.

show abstract

Section: Introductionmentioning

confidence: 99%

RNA Interference in Pigs: Comparison of RNAi Test Systems and Expression Vectors

et al. 2010

View full text Add to dashboard Cite

show abstract

“…Unfortunately, however, somatic cells have a limited life span in culture (Denning et al, 2001). To achieve a targeted genetic modification, the cells must survive through cell isolation and expansion, the targeting process and the preparation for NT, a process that requires about 45 population doublings (Clark et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

UP1 Extends Life of Primary Porcine Fetal Fibroblasts in Culture

Mir

Tanner

Chowdhary

et al. 2003

Cloning and Stem Cells

View full text Add to dashboard Cite

Genetic modification of somatic cell nuclei and subsequent nuclear transfer has opened an opportunity to create gene-targeted animals. However, somatic cells have a limited life span in culture and it is not possible to introduce precise genetic changes in both alleles in this narrow time window. To increase the life span of somatic cell in culture, both genetic and chemical approaches have been tried with varying success. Here, we report the effect of two anti-oxidants, glutathione and n-t-butyl hydroxylamine, and of the expression of UP1, a shortened derivative of heterogeneous nuclear riboprotein (hnRNP)A1, on the life extension of primary porcine fibroblasts in culture. Under our experimental conditions, the use of anti-oxidants did not result in any prolongation of the life span. In contrast, UP1 expression increased the life span significantly. While most control cells stopped growing by PDL 20, and none survived beyond PDL 35, 100% of UP1-expressing clones reached PDL50, and 40% made it to PDL65. The five UP1-expressing clones were karyotyped at PDL 50. While all of them had a range of numerical chromosomal abnormalities, two clones retained 30-40% normal cells, all the cells in other three clones had abnormal chromosome numbers. Thus, expression of UP1 may be useful in extending the life span of somatic cells in culture. This, in turn, will facilitate the process of gene targeting in this cell type.

show abstract

“…At present, the perspective of the propagation of transgenic mammalian species with knock-out alleles of gene encoding pathogenic (multimer) isoform of prion protein (PrP Sc ) is the most attractive aspect of combined technologies of targeted (locus-specific) mutagenesis and somatic cell cloning for animal breeding and veterinary medicine (Wrathall 2000, Denning et al 2001a (Fig. 1).…”

Section: Perspectives Of Somatic Cell Cloning and Transgenesis Of Farmentioning

confidence: 99%

“…However, significant challenges, such as establishment of somatic cell gene targeting techniques, must be overcome before the technology can be applied routinely. Denning et al (2001a) achieved targeted deletion involving single alleles of both the α-1,3-galactosyltransferase gene and prion protein gene at the GGTA1 and PRNP counterpart loci in primary fetal fibroblast cells from livestock (sheep and pigs). They place particular emphasis on the characteristics of the proliferative activity for the primary cell cultures of nuclear donor fibroblasts, because these are key to determining success in somatic cell cloning.…”

Section: Perspectives Of Somatic Cell Cloning and Transgenesis Of Farmentioning

confidence: 99%

The possibilities of practical application of transgenic mammalian species generated by somatic cell cloning in pharmacology, veterinary medicine and xenotransplantology

Samiec

Skrzyszowska

2011

Polish Journal of Veterinary Sciences

View full text Add to dashboard Cite

Cloning of genetically-modified mammals to produce: 1) novel animal bioreactors expressing human genes in rens, urinary bladder and the male accessory sex glands, as well as 2) porcine organs suitable in pig-to-human xenotransplantology, could offer new advantages for biomedical purposes. So too does the generation and/or multiplication of genetically-engineered cloned animals in order to produce: 3) physiologically-relevant animal models of serious monogenic human diseases and 4) prion disease-resistant small as well as large animals (i.e., rodents, ruminants). The basic purpose of this paper is to overview current knowledge deciphering the possibilities of using transgenic specimens created by somatic cell nuclear transfer in medical pharmacology, veterinary medicine, agriculture, transplantational medicine and immunology.

show abstract

Gene Targeting in Primary Fetal Fibroblasts from Sheep and Pig

Cited by 64 publications

References 27 publications

RNA Interference in Pigs: Comparison of RNAi Test Systems and Expression Vectors

RNA Interference in Pigs: Comparison of RNAi Test Systems and Expression Vectors

UP1 Extends Life of Primary Porcine Fetal Fibroblasts in Culture

The possibilities of practical application of transgenic mammalian species generated by somatic cell cloning in pharmacology, veterinary medicine and xenotransplantology

Contact Info

Product

Resources

About