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Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

Chang K, Qian J, Jiang M, Liu YH, Wu MC, Chen CD, Lai CK, Lo HL, Hsiao CT, Brown L, Bolen J, Huang HI, Ho PY, Shih PY, Yao CW, Lin WJ, Chen CH, Wu FY, Lin YJ, Xu J, Wang K - BMC Biotechnol. (2002)

Bottom Line: Transgenic animals have become valuable tools for both research and applied purposes.Furthermore, expression of the transgene is demonstrated in 61% (35/57) of transgenic pigs (F0 generation).Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.

View Article: PubMed Central - HTML - PubMed

Affiliation: BioAgri Corporation-Taiwan Branch, Fl. 8-8, No. 8, Song-Chiang Rd., Taipei, Taiwan. kchang@bioagricorp.com.tw

ABSTRACT

Background: Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals.

Results: The linker protein, a monoclonal antibody (mAb C), is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57) of transgenic pigs (F0 generation).

Conclusions: Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.

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Genotype analysis of transgenic mice. Southern blot analysis of transgenic mice from F1 generation. The genomic DNA was isolated from 6-week-old mouse-tails and digested with Bgl I. The 1.3 kb Bgl I fragment of pSEAP-2 control DNA was used as a probe. The numbers in bold indicate positive detection on the blot.
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Figure 7: Genotype analysis of transgenic mice. Southern blot analysis of transgenic mice from F1 generation. The genomic DNA was isolated from 6-week-old mouse-tails and digested with Bgl I. The 1.3 kb Bgl I fragment of pSEAP-2 control DNA was used as a probe. The numbers in bold indicate positive detection on the blot.

Mentions: Generating transgenic mice by microinjection has been widely used in studies of gene function and regulation. To see if our LB-SMGT method can also be used in mice, FVB/N mouse sperm was collected by epididymal dissection and mixed with mAb C to form a sperm-mAb complex, then SalI linearized pSEAP-2 control DNA was added and in vitro fertilizations were performed. Initial screening by PCR showed 33% of the 10-day-old embryos carried the transgene (data not shown). Forty-seven F0 pups were born but no hybridization signal was detected in their tail samples by Southern blot. However, four (33%) transmitted transgenic mouse lines (F1) were found after twelve F0 generation animals were randomly selected to mate with wild type mice (Fig. 7). We also noticed that one out of five offspring from F0 #38 showed multiple hybridization bands (about 1 and 2.6 kb) indicating DNA rearrangement (Fig. 7). This may be a higher frequency of mosaicism than most published results in FVB/N mice produced by the microinjection method. In summary, our studies show that in the FVB/N inbred mice, LB-SMGT is a viable method for generating transgenic mice, although we observed a low fertilization rate (FVB/N is known as a difficult strain for IVF but is widely used for microinjection), few founders, and a high frequency of mosaicism. We may be able to improve the fertilization rate and simplify the method by using alternatives to in vitro fertilization such as oviduct, uterine, or artificial insemination. Since mAb C bound to all tested sperm from various species, LB-SMGT should be applicable with different strains of mice as well as with other rodents such as the rat.


Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

Chang K, Qian J, Jiang M, Liu YH, Wu MC, Chen CD, Lai CK, Lo HL, Hsiao CT, Brown L, Bolen J, Huang HI, Ho PY, Shih PY, Yao CW, Lin WJ, Chen CH, Wu FY, Lin YJ, Xu J, Wang K - BMC Biotechnol. (2002)

Genotype analysis of transgenic mice. Southern blot analysis of transgenic mice from F1 generation. The genomic DNA was isolated from 6-week-old mouse-tails and digested with Bgl I. The 1.3 kb Bgl I fragment of pSEAP-2 control DNA was used as a probe. The numbers in bold indicate positive detection on the blot.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC113740&req=5

Figure 7: Genotype analysis of transgenic mice. Southern blot analysis of transgenic mice from F1 generation. The genomic DNA was isolated from 6-week-old mouse-tails and digested with Bgl I. The 1.3 kb Bgl I fragment of pSEAP-2 control DNA was used as a probe. The numbers in bold indicate positive detection on the blot.
Mentions: Generating transgenic mice by microinjection has been widely used in studies of gene function and regulation. To see if our LB-SMGT method can also be used in mice, FVB/N mouse sperm was collected by epididymal dissection and mixed with mAb C to form a sperm-mAb complex, then SalI linearized pSEAP-2 control DNA was added and in vitro fertilizations were performed. Initial screening by PCR showed 33% of the 10-day-old embryos carried the transgene (data not shown). Forty-seven F0 pups were born but no hybridization signal was detected in their tail samples by Southern blot. However, four (33%) transmitted transgenic mouse lines (F1) were found after twelve F0 generation animals were randomly selected to mate with wild type mice (Fig. 7). We also noticed that one out of five offspring from F0 #38 showed multiple hybridization bands (about 1 and 2.6 kb) indicating DNA rearrangement (Fig. 7). This may be a higher frequency of mosaicism than most published results in FVB/N mice produced by the microinjection method. In summary, our studies show that in the FVB/N inbred mice, LB-SMGT is a viable method for generating transgenic mice, although we observed a low fertilization rate (FVB/N is known as a difficult strain for IVF but is widely used for microinjection), few founders, and a high frequency of mosaicism. We may be able to improve the fertilization rate and simplify the method by using alternatives to in vitro fertilization such as oviduct, uterine, or artificial insemination. Since mAb C bound to all tested sperm from various species, LB-SMGT should be applicable with different strains of mice as well as with other rodents such as the rat.

Bottom Line: Transgenic animals have become valuable tools for both research and applied purposes.Furthermore, expression of the transgene is demonstrated in 61% (35/57) of transgenic pigs (F0 generation).Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.

View Article: PubMed Central - HTML - PubMed

Affiliation: BioAgri Corporation-Taiwan Branch, Fl. 8-8, No. 8, Song-Chiang Rd., Taipei, Taiwan. kchang@bioagricorp.com.tw

ABSTRACT

Background: Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals.

Results: The linker protein, a monoclonal antibody (mAb C), is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57) of transgenic pigs (F0 generation).

Conclusions: Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.

Show MeSH
Related in: MedlinePlus