Targeted Genome Editing by Recombinant Adeno-Associated Virus (rAAV) Vectors for Generating Genetically Modified Pigs

Luo, Yonglun; Kofod-Olsen, Emil; Christensen, Rikke; Sørensen, Charlotte Brandt; Bolund, Lars

doi:10.1016/j.jgg.2012.05.004

Cited by 12 publications

(8 citation statements)

References 54 publications

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“…In contrast to conventional random transgene integration, the transposase-catalyzed DNA integration preferentially resulted in monomeric integrations in transcriptionally permissive loci of the genome. Together with more accurate whole genome data and highly specific designed DNA modifying enzymes [59], precision genetic modifications became feasible in the pig genome [21,25,70]. It is anticipated that authentic pluripotent cells of the pig will be generated in the near future.…”

Section: Discussionmentioning

confidence: 99%

Assessment of Fecundity and Germ Line Transmission in Two Transgenic Pig Lines Produced by Sleeping Beauty Transposition

Garrels

Holler

Cleve

et al. 2012

Genes

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Recently, we described a simplified injection method for producing transgenic pigs using a non-autonomous Sleeping Beauty transposon system. The founder animals showed ubiquitous expression of the Venus fluorophore in almost all cell types. To assess, whether expression of the reporter fluorophore affects animal welfare or fecundity, we analyzed reproductive parameters of two founder boars, germ line transmission, and organ and cell specific transgene expression in animals of the F1 and F2 generation. Molecular analysis of ejaculated sperm cells suggested three monomeric integrations of the Venus transposon in both founders. To test germ line transmission of the three monomeric transposon integrations, wild-type sows were artificially inseminated. The offspring were nursed to sexual maturity and hemizygous lines were established. A clear segregation of the monomeric transposons following the Mendelian rules was observed in the F1 and F2 offspring. Apparently, almost all somatic cells, as well as oocytes and spermatozoa, expressed the Venus fluorophore at cell-type specific levels. No detrimental effects of Venus expression on animal health or fecundity were found. Importantly, all hemizygous lines expressed the fluorophore in comparable levels, and no case of transgene silencing or variegated expression was found after germ line transmission, suggesting that the insertions occurred at transcriptionally permissive loci. The results show that Sleeping Beauty transposase-catalyzed transposition is a promising approach for stable genetic modification of the pig genome.

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Section: Discussionmentioning

confidence: 99%

Assessment of Fecundity and Germ Line Transmission in Two Transgenic Pig Lines Produced by Sleeping Beauty Transposition

Garrels

Holler

Cleve

et al. 2012

Genes

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“…AAV vectors are often used for gene targeting experiments in mammalian cells also in combination with zinc finger technology [15,32,33]. Several studies indicate that AAV mediated gene targeting is affected by homologous recombination genes and that the AAV integration can be dependent on non-homologous end joining [34-37]; however, to our knowledge no comparative study has been performed to understand the role of ITRs and Rep proteins in gene targeting and random integration.…”

Section: Resultsmentioning

confidence: 99%

Inverted terminal repeats of adeno-associated virus decrease random integration of a gene targeting fragment in Saccharomyces cerevisiae

Galli

Cervelli

2014

BMC Mol Biol

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BackgroundHomologous recombination mediated gene targeting is still too inefficient to be applied extensively in genomics and gene therapy. Although sequence-specific nucleases could greatly stimulate gene targeting efficiency, the off-target cleavage sites of these nucleases highlighted the risk of this strategy. Adeno-associated virus (AAV)-based vectors are used for specific gene knockouts, since several studies indicate that these vectors are able to induce site-specific genome alterations at high frequency. Since each targeted event is accompanied by at least ten random integration events, increasing our knowledge regarding the mechanisms behind these events is necessary in order to understand the potential of AAV-mediated gene targeting for therapy application. Moreover, the role of AAV regulatory proteins (Rep) and inverted terminal repeated sequences (ITRs) in random and homologous integration is not completely known. In this study, we used the yeast Saccharomyces cerevisiae as a genetic model system to evaluate whether the presence of ITRs in the integrating plasmid has an effect on gene targeting and random integration.ResultsWe have shown that the presence of ITRs flanking a gene targeting vector containing homology to its genomic target decreased the frequency of random integration, leading to an increase in the gene targeting/random integration ratio. On the other hand, the expression of Rep proteins, which produce a nick in the ITR, significantly increased non-homologous integration of a DNA fragment sharing no homology to the genome, but had no effect on gene targeting or random integration when the DNA fragment shared homology with the genome. Molecular analysis showed that ITRs are frequently conserved in the random integrants, and that they induce rearrangements.ConclusionsOur results indicate that ITRs may be a useful tool for decreasing random integration, and consequently favor homologous gene targeting.

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“…The "ras gene" is the most widely known and most commonly studied of these genes. After mutation, the ras gene initiates division and cancer formation in cells with the "ras protein" it forms (Luo et al, 2012).…”

Section: Cancermentioning

confidence: 99%

Gene Delivery Systems: Recent Progress in Viral and Non-Viral Therapy

Cevher¹,

Sezer²,

Çağlar³

2012

Recent Advances in Novel Drug Carrier Systems

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Targeted Genome Editing by Recombinant Adeno-Associated Virus (rAAV) Vectors for Generating Genetically Modified Pigs

Cited by 12 publications

References 54 publications

Assessment of Fecundity and Germ Line Transmission in Two Transgenic Pig Lines Produced by Sleeping Beauty Transposition

Assessment of Fecundity and Germ Line Transmission in Two Transgenic Pig Lines Produced by Sleeping Beauty Transposition

Inverted terminal repeats of adeno-associated virus decrease random integration of a gene targeting fragment in Saccharomyces cerevisiae

Gene Delivery Systems: Recent Progress in Viral and Non-Viral Therapy

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