Active integration: new strategies for transgenesis

Shinohara, Eric T.; Kaminski, Joseph; Segal, David J.; Pelczar, Pawel; Kolhe, Ravindra; Ryan, Thomas M.; Coates, Craig J.; Fraser, Malcolm J.; Handler, Alfred M.; Yanagimachi, Ryuzo; Moisyadi, Stefan

doi:10.1007/s11248-007-9077-z

Cited by 42 publications

(26 citation statements)

References 32 publications

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“…Recently, a new transgeneic model has been developed, in which ICSI-SMGT is combined with recombinase and transposase to increase the efficiency of the transmission of the exogenous gene. This technique has been denominated 'active transgenesis' (Shinohara et al 2007). We investigated the possibility of exploiting the endogenous properties of recombinase proteins that allow them to associate with, bind, and recombine DNA, and their compatibility with IVF and ICSI-SMGT techniques, in order to generate transgenic pigs.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, Shinohara et al (2007) affirmed that one of the problems of active transgenesis in mice is the high level of mosaicism. According to Chan et al (1999), the process of integration in the embryos that express GFP in 100% of the blastomeres probably occurred in the first cellular cycle, the pronuclear state before the replication of the DNA.…”

Section: Recombinase-mediated Pig Transgenesismentioning

confidence: 99%

“…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. In 'active transgenesis', recombinases or transposases are injected into oocytes to increase the efficiency of transgene integration into the genome (Shinohara et al 2007). …”

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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Section: Discussionmentioning

confidence: 99%

Section: Recombinase-mediated Pig Transgenesismentioning

confidence: 99%

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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“…To develop a pB system that is more suitable for transgenesis and gene therapy experiments, helper-independent single plasmids were constructed, containing both the donor and helper elements of the pB transposition system in the same circular construct, similar to plasmids previously described (20,21). This cis-acting plasmid configuration ensures that both components, transposon, and transposase, are delivered simultaneously to the nucleus and hence should result in an improved transposition Author contributions: J.U., J.M.K., R.Y., and S.M.…”

mentioning

confidence: 99%

Helper-independent piggyBac plasmids for gene delivery approaches: Strategies for avoiding potential genotoxic effects

Urschitz

Kawasumi

Owens

et al. 2010

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

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Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flanked by terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.

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“…However, the low rate of GFP-positive pups indicated a lethal shortcoming. To improve the success rate, several original plasmids and constructs have been designed to combine the transgene with the spermatozoa more easily (Kaneko et al 2005, Moreira et al 2005, Shinohara et al 2007). However, there have been no advances in treating the spermatozoa.…”

Section: Advantages Of Naoh-treated Spermatozoa and Their Future Applmentioning

confidence: 99%

Production of normal mice from spermatozoa denatured with high alkali treatment before ICSI

Li¹,

Mizutani²,

Ono³

et al. 2009

Reproduction

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In mammals, ICSI is now a very important tool for both assisted reproductive technology and studying the mechanisms of fertilization. In the latter experiments, it is important to use spermatozoa that have lost their oocyte activation capacity but still retain their developmental potential. In this study, we used high-concentration NaOH to remove oocyte activation potential from spermatozoa, and examined whether normal offspring could be generated from these spermatozoa after ICSI. The spermatozoa were treated with different concentrations of NaOH (1-100 mM) for 1 h and then neutralized with equal amounts of same concentration of HCl. In 10 mM NaOHtreated spermatozoa, the cell membrane was broken and most of them failed to activate oocytes after their injection into the oocytes. However, these spermatozoa did not show strong damage, and after artificial activation with SrCl 2 , all of the zygotes were judged as normal by immunostaining to check the methylation status of histone H3 lysine 9, low chromosome damage by karyotype assay and staining with DNA double-strand breaks marker, gH2AX. Moreover, after transferring those embryos into recipient females, 106 (36.7%) live and healthy offspring were delivered, which is similar to the rate in the fresh control group. By contrast, spermatozoa treated with lower NaOH concentrations retained their oocyte activation capacity and those treated with higher concentrations lost their developmental potential. This suggests that 10 mM NaOH for 1 h is the best treatment to completely destroy the cell membrane and activation capacity of spermatozoa without injuring their developmental potential.

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Active integration: new strategies for transgenesis

Cited by 42 publications

References 32 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

Helper-independent piggyBac plasmids for gene delivery approaches: Strategies for avoiding potential genotoxic effects

Production of normal mice from spermatozoa denatured with high alkali treatment before ICSI

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