Transgenic mouse offspring generated by ROSI |
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| Authors: | Pedro MOREIRA Serafín PéREZ-CEREZALES Ricardo LAGUNA Raúl FERNáNDEZ-GONZALEZ Belén Pintado SANJUANBENITO Alfonso GUTIéRREZ-ADáN |
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| Institution: | 1)Departamento de Reproducción Animal, INIA, Madrid 28040, Spain;2)Servicio de Transgénesis, CNB-CBMSO CSIC, UAM, Madrid 28049, Spain |
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| Abstract: | The production of transgenic animals is an important tool for experimental and applied biology. Over the
years, many approaches for the production of transgenic animals have been tried, including pronuclear
microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer
and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and
we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid
nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV
immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained
carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by
pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer
embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and
fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by
ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced
by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate
that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an
exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature
sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures
for transgene delivery into embryos or reconstituted oocytes. |
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| Keywords: | Intracytoplasmic sperm injection (ICSI) Round spermatid nucleus injection (ROSI) Transgenesis |
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