Factors affecting production of transgenic rats by ICSI-mediated DNA transfer: Effects of sonication and freeze-thawing of spermatozoa, rat strains for sperm and oocyte donors, and different constructs of exogenous DNA

Hirabayashi, Masumi; Kato, Megumi; Ishikawa, Ayako; Kaneko, Ryosuke; Yagi, Takeshi; Hochi, Shinichi

doi:10.1002/mrd.20223

Cited by 39 publications

(51 citation statements)

References 27 publications

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“…This result agreed well with our previous report that ICSI after exposure to 5 ng/µl DNA resulted in no development beyond the 4-cell stage in vitro [7]. However, even with the protection of RecA-coating, there was no EGFP-carrying transgenic rat among the 26 newborn offspring produced by this procedure.…”

supporting

confidence: 93%

“…In general, the proportion of microinjected zygotes developing into transgenic offspring is <5% in rodents [1,5], and <1% in large domestic species [17,22]. Another approach to production of transgenic animals is intracytoplasmic sperm injection (ICSI)-mediated transgenesis (ICSI-Tg) [16], in which <7% of inseminated oocytes develop into transgenic mice [16,20] and rats [7,9]. Maga et al [11] recently reported that recombinase-A protein (RecA)-coated ssDNA was more likely to be integrated into goat and pig genomes after pronuclear microinjection (6-and 10-fold higher, respectively).…”

mentioning

confidence: 99%

“…The expression of EGFP DNA in the offspring was examined by fluorescence under 480 nm UV light. The integration of the DNA into genome was determined in the ear tissue of the offspring by PCR analysis [6,7,9], with a primer set for EGFP (EGFPF: 5'-GTT CAG CGT GTC CGG CGA-3', EGFPR: 5'-GCG GTC ACG AAC TCC AGC-3') and with a primer set for OAMB (OAMBF: 5'-ATG AAT GAA ACA GAG TGC GAG GAT C-3', OAMBR: 5'-ATG GTT TCC CTT TGG ATA GGT CGG C-3').…”

mentioning

confidence: 99%

“…In Experiment 2, ICSI-Tg was performed using a protocol reported previously [7,9]. Briefly, a suspension of cauda epididymal spermatozoa was treated for 10 s using a 10% power output from an ultrasonic cell disruptor (Sonifier250, Branson, Danbury, CT, USA), and was then cryopreserved in liquid nitrogen until use.…”

mentioning

confidence: 99%

“…Kaneko et al [8] used 5 to 40 ng/µl EGFP DNA in the recombinase-mediated mouse transgenesis, with low cleavage rates (26 to 36%) but high offspring rates (28-67%). The final concentration of EGFP DNA in rat ICSI-Tg (0.1 ng/µl) was determined based on our previous study using the same DNA construct [7]. Since a protective action of RecA protein against the adverse effect of EGFP DNA on development of rat embryos was expected, an additional ICSI-Tg trial using ssEGFP/RecA at a final concentration of 5 ng/µl was attempted.…”

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confidence: 99%

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No Effect of Recombinase-Mediated DNA Transfer on Production Efficiency of Transgenic Rats

et al. 2006

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It was reported that recombinase-Although pronuclear DNA microinjection is the most convenient approach to produce transgenic animals, the low integration of exogenous DNA into genomes is still an obstacle to the widening of the transgenic technology. In general, the proportion of microinjected zygotes developing into transgenic offspring is <5% in rodents [1,5], and <1% in large domestic species [17,22]. Another approach to production of transgenic animals is intracytoplasmic sperm injection (ICSI)-mediated transgenesis (ICSI-Tg) [16], in which <7% of inseminated oocytes develop into transgenic mice [16,20] and rats [7,9]. Maga et al. [11] recently reported that recombinase-A protein (RecA)-coated

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confidence: 93%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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No Effect of Recombinase-Mediated DNA Transfer on Production Efficiency of Transgenic Rats

et al. 2006

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Efficient and simple production of transgenic mice and rabbits using the new DMSO‐sperm mediated exogenous DNA transfer method

Shen

Pan³

et al. 2006

Molecular Reproduction Devel

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A high efficient and simple transgenic technology on mice and rabbits to transfect spermatozoa with exogenous DNA/DMSO complex to obtain transgenic offspring, which is namely called DMSO-sperm mediated gene transfer (SMGT). Mouse sperm could be either directly transfected via injection into testis or cultured in vitro with the plasmed DNA containing the enhanced green fluorescent protein (EGFP) that could be expressed in the embryos and offspring. Then, 36 living transgenic rabbits were produced using the same technology, and the transgenic ratio of 56.3% was detected using PCR and Southern blot. As the controls, the transgenic ratios of 39.6% and 47.8% have also been tested using the liposomes mediated technology of Tfx-50 Reagent or Lipefectamin-2000, respectively. The results show that the female transgenic rabbits, as the mammary gland bioreactor models, could express the human tissue plasminogen activator mutant (htPAm) in their mammary cells when they are adult.

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Characterization of the ICSI‐mediated gene transfer method in the production of transgenic pigs

Umeyama

Saitô

Kurome

et al. 2011

Molecular Reproduction Devel

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Understanding the behavior of transgenes introduced into oocytes or embryos is essential for evaluating the methodologies for transgenic animal production. We investigated the expression pattern of a transgene transferred to porcine eggs by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) or pronuclear microinjection (PN injection). The introduction of the EGFP gene by ICSI-MGT yielded significantly more embryos with non-mosaic transgene expression (P < 0.01). In the ICSI-MGT group, 61.5% (24/39) of the embryos were EGFP-positive in all their component blastomeres at the morula stage, while fewer than 10% of such embryos were EGFP-positive in the PN-injection group. Using three types of transgenes, ranging from 3.0 to 7.5 kb in size, we confirmed that approximately one in four fetuses obtained by ICSI-MGT was transgenic, suggesting that ICSI-MGT is a practical method for transgenic pig production. Southern blot analysis of 12 transgenic fetuses produced by ICSI-MGT revealed that the number of integrated transgene copies varied from 1 to 300, with no correlation between transgene size and the number of integrated copies. Fluorescence in situ hybridization analysis revealed that the transgenes were randomly integrated into a single site on the host chromosomes. Together, these data indicate that multiple-copy, single-site integration of a transgene is the primary outcome of ICSI-MGT in the pig and that ICSI-MGT is less likely than PN injection to cause transgene integration in a mosaic manner.

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Factors affecting production of transgenic rats by ICSI-mediated DNA transfer: Effects of sonication and freeze-thawing of spermatozoa, rat strains for sperm and oocyte donors, and different constructs of exogenous DNA

Cited by 39 publications

References 27 publications

No Effect of Recombinase-Mediated DNA Transfer on Production Efficiency of Transgenic Rats

No Effect of Recombinase-Mediated DNA Transfer on Production Efficiency of Transgenic Rats

Efficient and simple production of transgenic mice and rabbits using the new DMSO‐sperm mediated exogenous DNA transfer method

Characterization of the ICSI‐mediated gene transfer method in the production of transgenic pigs

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