Evidence for Repeat-Induced Gene Silencing in Cultured Mammalian Cells: Inactivation of Tandem Repeats of Transfected Genes

McBurney, Michael W.; Mai, Tina; Yang, Xiaofeng; Jardine, Karen

doi:10.1006/excr.2001.5443

Cited by 82 publications

(59 citation statements)

References 33 publications

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“…That suggests that insertion of an exogenous gene may be more tolerable than silencing an endogenous one by an organism. Not all the functional deficiencies of endogenous genes can be compensated for by other genes, whereas transgene silencing is a prevalent problem, especially when genes integrate as multicopy transgene arrays (Garrick et al, 1998;McBurney et al, 2002;Muskens et al, 2000). Although having low efficiency, the live offspring resulting from KO can be bred with traditional reproduction.…”

Section: Discussionmentioning

confidence: 99%

Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos

Liu

Dou

Xiang

et al. 2015

Cellular Reprogramming

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Data analysis in somatic cell nuclear transfer (SCNT) research is usually limited to several hundreds or thousands of reconstructed embryos. Here, we report mass results obtained with an established and consistent porcine SCNT system (handmade cloning [HMC]). During the experimental period, 228,230 reconstructed embryos and 82,969 blastocysts were produced. After being transferred into 656 recipients, 1070 piglets were obtained. First, the effects of different types of donor cells, including fetal fibroblasts (FFs), adult fibroblasts (AFs), adult preadipocytes (APs), and adult blood mesenchymal (BM) cells, were investigated on the further in vitro and in vivo development. Compared to adult donor cells (AFs, APs, BM cells, respectively), FF cells resulted in a lower blastocyst/reconstructed embryo rate (30.38% vs. 37.94%, 34.65%, and 34.87%, respectively), but a higher overall efficiency on the number of piglets born alive per total blastocysts transferred (1.50% vs. 0.86%, 1.03%, and 0.91%, respectively) and a lower rate of developmental abnormalities (10.87% vs. 56.57%, 24.39%, and 51.85%, respectively). Second, recloning was performed with cloned adult fibroblasts (CAFs) and cloned fetal fibroblasts (CFFs). When CAFs were used as the nuclear donor, fewer developmental abnormalities and higher overall efficiency were observed compared to AFs (56.57% vs. 28.13% and 0.86% vs.

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Section: Discussionmentioning

confidence: 99%

Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos

Liu

Dou

Xiang

et al. 2015

Cellular Reprogramming

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“…The expression of LacZ varied widely among the three founder lines, although ubiquitous, non-selective expression was not observed in any of the lines. The variability of expression suggests that there may be significant silencing of transgene expression depending on the insertion site of the recombinant sequence, a commonly observed phenomenon in neural systems 15 (this was especially true for the DsRed portion of the gene, which appeared to be completely silenced in all three transgenic lines). Animals from CCID line B showed the widest expression of LacZ, which was essentially identical to endogenous CCK gene expression (Figure 7a and c), suggesting that they may be suitable for use in generating CCK-selective knockouts when crossed with animals containing floxed transgenes.…”

Section: Transgene Expression For Fluorescence-guided Electrophysiolomentioning

confidence: 96%

Identification of cell-type-specific promoters within the brain using lentiviral vectors

et al. 2007

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The development of cell-type-specific mini-promoters for genetic studies is complicated by a number of issues. Here, we describe a general method for the relatively rapid screening of specific promoter activity in cell culture, in acute brain slice preparations and in vivo. Specifically, we examine the activity of an B3 kb promoter region from the neuroactive peptide cholecystokinin (CCK) compared to the commonly used cytomegalovirus promoter. We find a high degree of cell-type selectivity in vivo using lentiviral approaches in rats and traditional transgenic approaches in mice. Appropriate colocalization of Cre-recombinase and CCK gene expression is found within the hippocampus, when the CCK promoter is driving either the expression of Cre-recombinase or green fluorescent protein. We also demonstrate fluorescent identification of CCK-positive interneurons that allows for cell-type-specific electrophysiologic studies in rats and mice. In conclusion, these studies identify a functional mini-promoter for the CCK gene and outline a novel and sensitive general method to test activity of selective promoters in vitro and in vivo. This approach may allow for the more rapid identification of specific promoters for use with transgenic animals, in genetically modified viruses, and in the design of targeted, therapeutic genedelivery systems.

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“…Increasing the number of transgene copies within a tandem array has been shown to correlate with decreased levels of transgene expression: this may be explained by the fact that integrated concatemers can incur increased methylation 68 and reduced histone acetylation. 69 Thus, illegitimate integration events can have dramatic repercussions on the content, organization, and functions of the recipient genome.…”

Section: Introductionmentioning

confidence: 99%

Illegitimate DNA integration in mammalian cells

2003

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Foreign DNA integration is one of the most widely exploited cellular processes in molecular biology. Its technical use permits us to alter a cellular genome by incorporating a fragment of foreign DNA into the chromosomal DNA. This process employs the cell's own endogenous DNA modification and repair machinery. Two main classes of integration mechanisms exist: those that draw on sequence similarity between the foreign and genomic sequences to carry out homology-directed modifications, and the nonhomologous or 'illegitimate' insertion of foreign DNA into the genome. Gene therapy procedures can result in illegitimate integration of introduced sequences and thus pose a risk of unforeseeable genomic alterations. The choice of insertion site, the degree to which the foreign DNA and endogenous locus are modified before or during integration, and the resulting impact on structure, expression, and stability of the genome are all factors of illegitimate DNA integration that must be considered, in particular when designing genetic therapies.

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Evidence for Repeat-Induced Gene Silencing in Cultured Mammalian Cells: Inactivation of Tandem Repeats of Transfected Genes

Cited by 82 publications

References 33 publications

Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos

Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos

Identification of cell-type-specific promoters within the brain using lentiviral vectors

Illegitimate DNA integration in mammalian cells

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