Factors affecting porcine sperm mediated gene transfer

García–Vázquez, Francisco Alberto; Ruiz, Salvador; Grullón, Luis Alberto; Ondiz, Aitor de; Gutiérrez‐Adán, Alfonso; Gadea, J.

doi:10.1016/j.rvsc.2010.09.015

Cited by 31 publications

(19 citation statements)

References 60 publications

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“…In conjunction with cold-and heatshock treatment, this method has significantly increased DNA-sperm binding in mouse and rabbit IVF (Kuznetsov et al 2000. However, no effect on DNA-sperm binding or sperm viability was found in pig (Garcia-Vazquez et al 2011). We also found no significant increase in DNA binding to DMSOtreated sperm.…”

Section: Discussionmentioning

confidence: 46%

“…Using sperm-mediated gene transfer (SMGT) in combination with IVF (SMGT-IVF), efficient production of transgenic mice was subsequently reported in 1989 (Lavitrano et al 1989). Since then, various contradictory reports on applying this technique to livestock were published (Gandolfi et al 1996, Garcia-Vazquez et al 2011. By combining SMGT with laparoscopic insemination or artificial insemination (AI; SMGT-AI), one laboratory has reported transgenic embryos and offspring in pigs (Lavitrano et al 2002, Manzini et al 2006.…”

Section: Introductionmentioning

confidence: 99%

“…However, transgene transmission appeared to be only transient (Giovannoni et al 2010). Efficiency and reproducibility of the technique was poor in other laboratories and species (Brinster et al 1989, Gandolfi et al 1996, Chan et al 2000, Garcia-Vazquez et al 2011, Pereyra-Bonnet et al 2011. As a consequence, SMGT has not become a widely and routinely used method for generating transgenic animals, despite its potential for simple, low-cost, scalable transgenesis with large transgenes (Osada et al 2005).…”

Section: Introductionmentioning

confidence: 99%

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Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Eghbalsaied¹,

Ghaedi²,

Laible³

et al. 2013

Reproduction

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Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (nZ96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (nZ751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68Z56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (nZ48) or GFP fluorescence (nZ94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.

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Section: Discussionmentioning

confidence: 46%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Eghbalsaied¹,

Ghaedi²,

Laible³

et al. 2013

Reproduction

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“…Only a few species are amenable to the micromanipulations necessary for efficient ICSI-Tr. For example, ICSI-Tr is inefficient in pigs, and therefore appears unsuitable for routine transgenic production (34)(35)(36). Lentiviral-mediated transgenesis, on the other hand, is very efficient in delivering the transgene to the host genome but results in multicopy insertions; as a consequence, only a few micromanipulated embryos reach term, probably due to lethal mutagenesis.…”

Section: Discussionmentioning

confidence: 99%

Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis

Marh

Stoytcheva

Urschitz

et al. 2012

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

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We have developed a unique method for mouse transgenesis. The transposase-enhanced pronuclear microinjection (PNI) technique described herein uses the hyperactive piggyBac transposase to insert a large transgene into the mouse genome. This procedure increased transgene integration efficiency by fivefold compared with conventional PNI or intracytoplasmic sperm injection-mediated transgenesis. Our data indicate that the transposase-enhanced PNI technique additionally requires fewer embryos to be microinjected than traditional methods to obtain transgenic animals. This transposase-mediated approach is also very efficient for single-cell embryo cytoplasmic injections, offering an easy-to-implement transgenesis method to the scientific community.transgene | genetically modified

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“…Sperm-mediated gene transfer (SMGT) is based on the ability of sperm cells to bind, internalize, and transport exogenous DNA into an oocyte during fertilization [10]. To date, successful SMGT has been reported for a variety of animal classes, including mammals, birds, fish, and insects [11][12][13].…”

Section: Introductionmentioning

confidence: 99%

Germline Transformation of the Silkworm Bombyx mori L. by Sperm-Mediated Gene Transfer1

Zhou¹,

Gong

Xue

et al. 2012

Biology of Reproduction

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The domesticated silkworm Bombyx mori L. has important roles in basic biological research and applied science. To explore the practical use of transgenic technology in agricultural silkworm varieties, we fused the neomycin-resistance gene (Neo(R)) and the green fluorescent protein gene (gfp) into the piggyBac-based transposon vector and transduced it into silkworms by sperm-mediated gene transfer (SMGT). Fluorescence observation indicated the positive rate of G0 egg-batches is 72.7%. After screening against the antibiotic G418, development of individual larvae in the same brood showed significant size differences. PCR detection indicated the existence of gfp and Neo(R) and confirmed the positive rate of transgenesis as 0.47%. Southern blot analysis confirmed the presence of the exogenous genes in the genome of G7 larvae. These results show that our strategy is practical and markedly improves the efficiency of SMGT.

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Factors affecting porcine sperm mediated gene transfer

Cited by 31 publications

References 60 publications

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis

Germline Transformation of the Silkworm Bombyx mori L. by Sperm-Mediated Gene Transfer1

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