Efficient and simple production of transgenic mice and rabbits using the new DMSO-sperm mediated exogenous DNA transfer method.
ABSTRACT 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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ABSTRACT: Simian virus 40 (SV40) adsorbs on rabbit spermatozoa but does not penetrate the cells, as indicated by the absence of radioactive material seen on autoradiography of spermatozoa exposed to [(3)H]thymidine-labeled SV40. In contrast, after exposure of spermatozoa to labeled SV40 DNA, radioactive material was found in the postacrosomal area of the spermatozoa. Furthermore, when spermatozoa exposed to SV40 DNA were fused with cells of the CV-1 line of African green monkey kidney cells, infectious SV40 was isolated. After uterine insemination of rabbits with spermatozoa infected with SV40 DNA, both unfertilized and one- and two-celled fertilized ova were obtained. When the fertilized ova were cocultivated with CV-1 cells, infectious virus was recovered. In contrast, CV-1 cells exposed to the unfertilized ova or to zonae pellucidae or polar bodies from the fertilized ova did not show a cytopathic effect. This report provides the first evidence that a heterologous genome can be incorporated into a mammalian spermatozoon and subsequently carried into an ovum during the process of fertilization.Proceedings of the National Academy of Sciences 03/1971; 68(2):353-7. · 9.74 Impact Factor
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ABSTRACT: Classical approaches for producing transgenic livestock require labor-intensive, time-consuming, and expensive methods with low efficiency of transgenic production. A promising approach for producing transgenic animals by using male stem cells was recently reported by Brinster and Zimmermann (1994; Proc Natl Acad Sci 91:11298-11302) and by Brinster and Avarbock (1994: Proc Natl Acad Sci USA 91:11303-11307). However, in order to apply this technique to producing transgenic animals, some difficulties have to be overcome. These include a satisfactory method for short-term in vitro culture for drug selection after transfection with exogenous DNA, and methods for the use of livestock such as pigs. We developed a new method for transferring foreign DNA into male germ cells. Mice and pigs were treated with busulfan, an alkylating agent, to destroy the developing male germ cells, and liposome/bacterial LacZ gene complexes were introduced into each seminiferous tubule by using a microinjection needle. As a control, lipofectin was dissolved in phosphate-buffered saline at a ratio of 1:1, and then injected into seminiferous tubules. In mice, 8.0-14.8% of seminiferous tubule expressed the introduced LacZ gene, and 7-13% of epididymal spermatozoa were confirmed as having foreign DNA by polymerase chain reaction. The liposome-injected testes were all negative for X-gal staining. These results indicate that some spermatozoa were successfully transformed in their early stages by liposome/DNA complexes. In pigs, foreign DNA was also incorporated efficiently into male germ cells, and 15.3-25.1% of the seminiferous tubules containing germ cells expressed the LacZ gene. The data suggest that these techniques can be used as a powerful tool for producing transgenic livestock.Molecular Reproduction and Development 05/1997; 46(4):515-26. · 2.81 Impact Factor
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ABSTRACT: Uptake of exogenous DNA by electroporated salmon sperm for gene transfer is being investigated. Our studies show that electroporated salmon sperm cells were more efficient and more reliable than untreated sperm in picking up exogenous DNA and subsequently transferring the DNA into salmon embryos. Indirect evidence suggest that some of the exogenous DNA was internalized in the sperm nuclei. The taken up DNA retained its integrity as demonstrated by PCR. The foreign DNA was detected in 15-month-old fish, and had a mosaic pattern of distribution. Integration of the foreign DNA occurred infrequently, and the expression of the foreign genes was poor. The potential of sperm-mediated gene transfer as a routine protocol for mass gene transfer in salmon will be dependent on the improvement of integration and expression of the foreign gene.Molecular Reproduction and Development 07/2000; 56(2 Suppl):285-8. · 2.81 Impact Factor