Stable and unstable transgene integration sites in the human genome: extinction of the Green Fluorescent Protein transgene in K562 cells

Migliaccio, Anna Rita; Bengra, Chikh; Ling, Jianhua; Pi, Wenhu; Li, Chunhua; Zeng, Shan; Keskintepe, Meral; Whitney, Barry; Sanchez, Massimo; Migliaccio, Giovanni; Tuan, Dorothy

doi:10.1016/s0378-1119(00)00353-x

Cited by 42 publications

(35 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Because GFP diffuses out of compromised protoplasts but they may have physical properties otherwise nearly indistinguishable from intact protoplasts, the number of cells with wild-type levels of fluorescence can be overestimated. To correct for this phenomenon, we used an approach common in animal systems by which cells are stained with propidium iodide to exclude necrotic cells from analysis, for example, Migliaccio et al (2000). Under conditions where debris is a problem, adding this step to existing plant cell flow cytometry protocols would enhance our ability to discriminate between expressing and nonexpressing subpopulations of cells.…”

Section: Discussionmentioning

confidence: 99%

The Rb7 Matrix Attachment Region Increases the Likelihood and Magnitude of Transgene Expression in Tobacco Cells: A Flow Cytometric Study

2005

View full text Add to dashboard Cite

Many studies in both plant and animal systems have shown that matrix attachment regions (MARs) can increase expression of transgenes in whole organisms or cells in culture. Because histochemical assays often indicate variegated transgene expression, a question arises: Do MARs increase transgene expression by increasing the percentage of cells expressing the transgene (likelihood), by increasing the level of expression in expressing cells (magnitude), or both? To address this question, we used flow cytometry to measure green fluorescent protein (GFP) expression in individual tobacco (Nicotiana tabacum) cells from lines transformed by Agrobacterium tumefaciens. We conclude that MAR-mediated overall increases in transgene expression involve both likelihood and magnitude. On average, cell lines transformed with the Rb7 MAR-containing vector expressed GFP at levels 2.0- to 3.7-fold higher than controls. MAR lines had fewer nonexpressing cells than control lines (10% versus 45%), and the magnitude of GFP expression in expressing cells was greater in MAR lines by 1.9- to 2.9-fold. We also show that flow cytometry measurements on cells from isogenic lines are consistent with those from populations of independently transformed cell lines. By obviating the need to establish isogenic lines, this use of flow cytometry could greatly simplify the evaluation of MARs or other sequence elements that affect transgene expression

show abstract

Section: Discussionmentioning

confidence: 99%

The Rb7 Matrix Attachment Region Increases the Likelihood and Magnitude of Transgene Expression in Tobacco Cells: A Flow Cytometric Study

2005

View full text Add to dashboard Cite

show abstract

“…CMV promoters can be quickly silenced by DNA methylation (40), whereas high and persistent levels of expression can be achieved in the CNS with rAAV using the CAG promoter (CMV enhancer, chicken β-actin hybrid promoter) and a Woodchuck hepatitis virus postranscriptional regulatory element (WPRE) (41,42). We therefore produced a second generation of rAAV using these elements.…”

Section: Competition Assays Show Preferential Repression Of Long Cagmentioning

confidence: 99%

Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice

Garriga-Canut

Agustín-Pavón

Herrmann

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

170

142

View full text Add to dashboard Cite

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by expanded CAG repeats in the huntingtin (HTT) gene. Although several palliative treatments are available, there is currently no cure and patients generally die 10-15 y after diagnosis. Several promising approaches for HD therapy are currently in development, including RNAi and antisense analogs. We developed a complementary strategy to test repression of mutant HTT with zinc finger proteins (ZFPs) in an HD model. We tested a "molecular tape measure" approach, using long artificial ZFP chains, designed to bind longer CAG repeats more strongly than shorter repeats. After optimization, stable ZFP expression in a model HD cell line reduced chromosomal expression of the mutant gene at both the protein and mRNA levels (95% and 78% reduction, respectively). This was achieved chromosomally in the context of endogenous mouse HTT genes, with variable CAG-repeat lengths. Shorter wild-type alleles, other genomic CAG-repeat genes, and neighboring genes were unaffected. In vivo, striatal adeno-associated virus viral delivery in R6/2 mice was efficient and revealed dose-dependent repression of mutant HTT in the brain (up to 60%). Furthermore, zinc finger repression was tested at several levels, resulting in protein aggregate reduction, reduced decline in rotarod performance, and alleviation of clasping in R6/2 mice, establishing a proof-of-principle for synthetic transcription factor repressors in the brain.

show abstract

“…54 Indeed, a cell population will often lose most of the unselected foreign DNA during the first weeks of expansion, while the selected DNA appears much more stable. 56,70 Many studies on SV40 transformed cell lines reported sustained instability involving the SV40 sequences and flanking cellular DNA upon continued culturing, 56,[71][72][73] suggesting that integrated structures are often unstable immediately after integration, and that this instability can persist in the absence of selection. While in some cases, tandem arrays of transgenes remain stable following integration, 41 other groups have observed homologous recombination between copies within the array, which lead to changes in structure, expression, and transgene copy number 74,75 (see also our unpublished results).…”

Section: Recipient Genomic Loci Are Often Unstable After Illegitimatementioning

confidence: 99%

Illegitimate DNA integration in mammalian cells

2003

View full text Add to dashboard Cite

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.

show abstract

Stable and unstable transgene integration sites in the human genome: extinction of the Green Fluorescent Protein transgene in K562 cells

Cited by 42 publications

References 32 publications

The Rb7 Matrix Attachment Region Increases the Likelihood and Magnitude of Transgene Expression in Tobacco Cells: A Flow Cytometric Study

The Rb7 Matrix Attachment Region Increases the Likelihood and Magnitude of Transgene Expression in Tobacco Cells: A Flow Cytometric Study

Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice

Illegitimate DNA integration in mammalian cells

Contact Info

Product

Resources

About