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Giraldo, Patricia; Montoliu, Lluı́s

doi:10.1023/a:1008918913249

Cited by 289 publications

(66 citation statements)

References 174 publications

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“…The development of BAC technology for generation of transgenic mice has alleviated this problem to a large extent. Coupled with the development of methodology to specifically mutate large DNA molecules (13), now termed recombineering (14,15), BACs are now the preferred choice to generate transgenic animals (16,17).…”

mentioning

confidence: 99%

RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells

Kittler

Pelletier

et al. 2005

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

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RNA interference (RNAi) is a widely used method for analysis of gene function in tissue culture cells. However, to date there has been no reliable method for testing the specificity of any particular RNAi experiment. The ideal experiment is to rescue the phenotype by expression of the target gene in a form refractory to RNAi. The transgene should be expressed at physiological levels and with its different splice variants. Here, we demonstrate that expression of murine bacterial artificial chromosomes in human cells provides a reliable method to create RNAi-resistant transgenes. This strategy should be applicable to all eukaryotes and should therefore be a standard technology for confirming the specificity of RNAi. We show that this technique can be extended to allow the creation of tagged transgenes, expressed at physiological levels, for the further study of gene function.off-target effects ͉ loss-of-function experiment ͉ interferon response ͉ gene knockdown

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mentioning

confidence: 99%

RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells

Kittler

Pelletier

et al. 2005

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

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“…To avoid these problems we opted for a large transgene (*210 kb) because we could legitimately expect it to contain both genes in their entirety, including the short-range (UTRs, introns) and long-range cis-regulatory elements required for correct spatio-temporal expression. The second advantage of the use of a BAC is that BACs are expected to be more resistant to position effects than smaller transgenes (Giraldo and Montoliu 2001;Gong et al 2003). Overall, we have confirmed the advantages linked to a larger transgene size, since we have detected inducible production of the boMx proteins in all organs of *90% (8/9) of the lines showing germinal insertion, whereas expression of the huMxA protein was previously demonstrated in only *13% (2/15) of the transgenic lines obtained (Pavlovic et al 1995).…”

Section: Discussionmentioning

confidence: 99%

Modulating mouse innate immunity to RNA viruses by expressing the Bos taurus Mx system

et al. 2009

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Mx proteins are interferon-induced members of the dynamin superfamily of large guanosine triphosphatases. These proteins have attracted much attention because some display antiviral activity against pathogenic RNA viruses, such as members of the orthomyxoviridae, bunyaviridae, and rhabdoviridae families. Among the diverse mammalian Mx proteins examined so far, we have recently demonstrated in vitro that the Bos taurus isoform 1 (boMx1) is endowed with exceptional anti-rabies-virus activity. This finding has prompted us to seek an appropriate in vivo model for confirming and evaluating gene therapy strategies. Using a BAC transgene, we have generated transgenic mouse lines expressing the antiviral boMx1 protein and boMx2 proteins under the control of their natural promoter and short- and long-range regulatory elements. Expressed boMx1 and boMx2 are correctly assembled, as deduced from mRNA sequencing and western blotting. Poly-I/C-subordinated expression of boMx1 was detected in various organs by immunohistochemistry, and transgenic lines were readily classified as high- or low-expression lines on the basis of tissue boMx1 concentrations measured by ELISA. Poly-I/C-induced Madin-Darby bovine kidney cells, bovine turbinate cells, and cultured cells from high-expression line of transgenic mice were found to contain about the same concentration of boMx1, suggesting that this protein is produced at near-physiological levels. Furthermore, insertion of the bovine Mx system rendered transgenic mice resistant to vesicular-stomatitis-virus-associated morbidity and mortality, and embryonic fibroblasts derived from high-expression transgenic mice were far less permissive to the virus. These results demonstrate that the Bos taurus Mx system is a powerful anti-VSV agent in vivo and suggest that the transgenic mouse lines generated here constitute a good model for studying in vivo the various antiviral functions-known and yet to be discovered-exerted by bovine Mx proteins, with priority emphasis on the antirabic function of boMx1.

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“…Random integration of the gene constructs may also result in varying, aberrant, or abolished transgene expression, because of effects of the adjacent chromatin overcoming the transgene's regulatory sequences. One possible means of avoiding these integration site-dependent effects is the transfer of large DNA constructs, which can form functionally independent chromatin domains [22]. The first successful example for this strategy in livestock was the generation of transgenic rabbits harboring yeast artificial chromosomes (YACs) [23].…”

Section: Pronuclear Dna Microinjectionmentioning

confidence: 99%

“…An increasing number of transgenic animals therefore carry gene constructs based on phage (PAC), bacterial (BAC), or yeast (YAC) artificial chromosomes [22]. For expression and replication, these large transgenes depend on integration into the host genome.…”

Section: Transgenes -Gene Constructsmentioning

confidence: 99%