Effect of sperm treatment on efficiency of EGFP-expressing porcine embryos produced by ICSI-SMGT

García-Vázquez, F. A.; García-Roselló, E.; Gutiérrez‐Adán, Alfonso; Gadea, J.

doi:10.1016/j.theriogenology.2009.04.006

Cited by 40 publications

(25 citation statements)

References 60 publications

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“…In this case, changes in the sperm membrane functionality might modulate the time of DNA binding; the populations of viable sperm attached to the DNA may determine success or failure in the production of transgenic animals using this method. Under our experimental conditions, we have confirmed that the cells with the most DNA bound are the dead cells or the cells with severe membrane alterations in boar spermatozoa (García-Vázquez et al 2009). …”

supporting

confidence: 73%

“…Damage to the sperm plasma membranes causes DNA to associate with important consequences for the transfer of DNA into oocytes (Perry et al 1999). However, sperm pretreatments that markedly disrupt the plasma membrane cause DNA fragmentation (Szczygiel et al 2003), or can damage the sperm nucleus and lead to lower efficiency of embryogenesis following ICSI (Kurome et al 2006, García-Vázquez et al 2009). It is also commonly found that transgenes integrated by passive transgenesis commonly form long concatameric arrays .…”

Section: F a García-vázquez And Othersmentioning

confidence: 99%

“…Semen was prepared according to the method described by Lavitrano et al (2002) and García-Vázquez et al (2009). Spermatozoa (10 8 cells/ml) were incubated with transgene (pEGFP) or RecA:ssDNA linearized EGFP transgene complexes (5 or 15 mg DNA/ml) in SFM with BSA at 16 8C.…”

Section: Semen Collection and Preparation Of Sperm For Dna Uptakementioning

confidence: 99%

“…Denuded oocytes were washed two times in supplemented DPBS medium and transferred to ICSI drops. ICSI was performed as described by García-Roselló et al (2006) and García-Vázquez et al (2009).…”

Section: Icsi With the Construct (Egfp)mentioning

confidence: 99%

“…Another innovation in SMGT has been the use of ICSI to deliver transgene-containing sperm cells directly into the egg, a process known as 'ICSI-mediated transgenesis' (ICSI-Tr) which was reported for the first time in mice (Perry et al 1999) and later developed in pigs , Pereyra-Bonnet et al 2008, García-Vázquez et al 2009, Wu et al 2009). These techniques (pronuclear injection and ICSI) are considered as passive methods for transgenesis (Shinohara et al 2007), which rely on the repair mechanisms of the host for transgene insertion.…”

Section: Introductionmentioning

confidence: 99%

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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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supporting

confidence: 73%

Section: F a García-vázquez And Othersmentioning

confidence: 99%

Section: Semen Collection and Preparation Of Sperm For Dna Uptakementioning

confidence: 99%

Section: Icsi With the Construct (Egfp)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

García–Vázquez¹,

Ruiz²,

Matás³

et al. 2010

Reproduction

View full text Add to dashboard Cite

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Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer (SMGT) technique

Cánovas

Gutiérrez‐Adán²,

Gadea

2010

Molecular Reproduction Devel

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Sperm mediated gene transfer (SMGT) could provide the opportunity to carry out transgenesis on a mass scale using spermatozoa as vectors for exogenous DNA. However, the efficiency of sperm-mediated DNA transfer is still questionable, and the mode of transmission to the egg has not yet been well understood. Our aim was to investigate the capacity of bovine spermatozoa to carry exogenous DNA and its relationship to sperm functionality. We studied these parameters using flow cytometry to measure viability (necrosis and apoptosis) and capacitation status, computer-assisted semen analysis (CASA) to measure motility parameters and in vitro fertilization (IVF) to assess fertilizing capacity. Furthermore, we studied the effect of capacitation status on interaction with exogenous DNA, and the role of heparin supplementation in this process. Bull spermatozoa showed a high capacity to bind DNA quickly and reached a maximum after 30 min, with approximately half of the DNA-bound spermatozoa being viable. Incubation with exogenous DNA induced a decrease in sperm viability and motility and increased the proportion of apoptotic cells, but did not affect the cleavage rate in IVF assay. Heparin increased high-lipid disorder and the number of sperm with DNA bound (viable and dead). In conclusion, this study shows that live spermatozoa can bind exogenous DNA with a slight negative effect in some parameters of sperm function that in our opinion, would not drastically compromise fertility.

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Sperm status and DNA dose play key roles in sperm/ICSI‐mediated gene transfer in caprine

Shadanloo

Najafi

Hosseini

et al. 2010

Molecular Reproduction Devel

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In relation to the growing recent interest in the establishment of sperm-mediated gene transfer (SMGT) technology as a convenient and effective method for the simple production of transgenic animals, in this study the possibility of using SMGT to produce transgenic caprine embryos was investigated for the first time. Buck sperm were directly incubated with different concentrations (0-500 ng) of pcDNA/his/Lac-Z plasmid and used for IVF or ICSI. Sperm used for ICSI were categorized into motile or live-immotile group before being injected into oocytes. In a separate experiment, dead sperm prepared by repeated freezing/thawing were used for DNA-incubation before ICSI. Sham injection was carried out by intracytoplasmic injection of approximately the same volume of media containing different doses of DNA using an ICSI needle. Transgene expression and transmission were detected by X-Gal staining and PCR analysis of developed embryos, respectively. A reasonable blastocyst rate was observed in all the groups. Only embryos in the sham group were negative for transgene transmission. Transgene expression was completely dependent on the delivery technique and status of sperm, and was only observed in the live-immotile and dead ICSI groups. The results of this study showed that the technique (IVF vs. ICSI vs. sham injection), sperm status (motile vs. live-immotile vs. dead) and to some extent DNA concentration affect embryo development, transgene transmission and expression.

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Effect of sperm treatment on efficiency of EGFP-expressing porcine embryos produced by ICSI-SMGT

Cited by 40 publications

References 60 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

Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer (SMGT) technique

Sperm status and DNA dose play key roles in sperm/ICSI‐mediated gene transfer in caprine

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