Protamine sulfate protects exogenous DNA against nuclease degradation but is unable to improve the efficiency of bovine sperm mediated transgenesis

Alderson, Jon; Wilson, B S Jonathan; Laible, Götz; Pfeffer, Peter; L'Huillier, P.J.

doi:10.1016/j.anireprosci.2005.03.005

Cited by 22 publications

(26 citation statements)

References 19 publications

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“…However, when 15 mg/ml of DNA were used in a RecA complex, the viability and motility significantly decreased. These results are in accordance with previous reports that confirmed that bovine and porcine spermatozoa can bind to exogenous DNA and maintain their motility (Rieth et al 2000, Alderson et al 2006, Kang et al 2008. Interestingly, one previous report (Chan 2000) described how transfected sperm had even higher percentages of total motility and progression when compared with controls.…”

Section: F a García-vázquez And Otherssupporting

confidence: 83%

Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA

García–Vázquez¹,

Ruiz²,

Matás³

et al. 2010

Reproduction

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Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) proteincoated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA-DNA complexes at 5 mg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT-ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.

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Section: F a García-vázquez And Otherssupporting

confidence: 83%

Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA

García–Vázquez¹,

Ruiz²,

Matás³

et al. 2010

Reproduction

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“…5 l heparin water solution (10 g/ml) was added into one of the two aliquots, the other was treated with the same volume (5 l) of pure water. The final solution was incubated at room temperature for 2 h so that the DNA could be released from the complexes [25]. The resulting product was evaluated by agarose gel electrophoresis.…”

Section: Dnase I Protection Assaymentioning

confidence: 99%

“…Recently, protamine and octaarginine have been used as DNA (or siRNA) condensing agents in many gene delivery systems [18][19][20][21][22]. They showed a strong nucleus-locating property [23,24] and the ability to protect DNA against DNase I in vitro [25]. But there are few reports on protamine as gene vector, due to its relatively low transfection efficiency.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and characterization of stearyl protamine and investigation of their complexes with DNA for gene delivery

Liu

Guo

et al. 2009

Colloids and Surfaces B: Biointerfaces

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“…These include electroporation (Gagne et al 1991, Rieth et al 2000, lipofection (Bachiller et al 1991, Hoelker et al 2007) and magnetofection (Kim et al 2010) as well as addition of DMSO (Kuznetsov & Kuznetsova 1995 or protamine (Alderson et al 2006). Irrespective of the transfection method, the mode of transferring exogenous DNA from the sperm into the oocyte via fertilisation is not well understood.…”

Section: Introductionmentioning

confidence: 99%

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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Protamine sulfate protects exogenous DNA against nuclease degradation but is unable to improve the efficiency of bovine sperm mediated transgenesis

Cited by 22 publications

References 19 publications

Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA

Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA

Synthesis and characterization of stearyl protamine and investigation of their complexes with DNA for gene delivery

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

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