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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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Related in: MedlinePlus

Southern blot analysis of transgenic pigs. a, Tail genomic DNA of group I and II F0 generation animals digested with Bgl I. b, and c, Ear genomic DNA from group I offspring (F1) digested with Bgl I. The numbers in bold indicate positive detection on the blot.
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Figure 3: Southern blot analysis of transgenic pigs. a, Tail genomic DNA of group I and II F0 generation animals digested with Bgl I. b, and c, Ear genomic DNA from group I offspring (F1) digested with Bgl I. The numbers in bold indicate positive detection on the blot.

Mentions: For this study, Duroc, Yorkshire, and Landrace female pigs (gilts) between 10 and 14 months old were selected for LB-SMGT experiments using surgical oviduct insemination (summary in Table 2). Forty-three offspring (#1–43) from 7 gilts (total 27 gilts were inseminated with a 26% pregnancy rate) were obtained in an initial experiment. To ensure detection of all transgenic samples, including those with a single copy of the transgene, we have examined genomic DNA from the tail region of piglets with Bgl I digestion, which has two internal restriction sites in the pSEAP-2 control DNA. Fig. 3a shows that ten out of seventy-five (13%) F0 generation piglets: 5 (not shown), 17, 26, 36, 40, 42, 43, 44, 48 and 64, had a 1.3 kb positive hybridization band. Animals 44–75 were obtained from a separate experiment (32 offspring from 8 gilts, total 30 gilts inseminated with a 28% pregnancy rate) to demonstrate reproducibility. Furthermore, analysis of sperm genomic DNA from a limited number of 8-month-old F0 generation animals (boars) showed that two out of eleven (18%), numbers 25 and 30, had a hybridization signal (data not shown).


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)

Southern blot analysis of transgenic pigs. a, Tail genomic DNA of group I and II F0 generation animals digested with Bgl I. b, and c, Ear genomic DNA from group I offspring (F1) digested with Bgl I. 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 3: Southern blot analysis of transgenic pigs. a, Tail genomic DNA of group I and II F0 generation animals digested with Bgl I. b, and c, Ear genomic DNA from group I offspring (F1) digested with Bgl I. The numbers in bold indicate positive detection on the blot.
Mentions: For this study, Duroc, Yorkshire, and Landrace female pigs (gilts) between 10 and 14 months old were selected for LB-SMGT experiments using surgical oviduct insemination (summary in Table 2). Forty-three offspring (#1–43) from 7 gilts (total 27 gilts were inseminated with a 26% pregnancy rate) were obtained in an initial experiment. To ensure detection of all transgenic samples, including those with a single copy of the transgene, we have examined genomic DNA from the tail region of piglets with Bgl I digestion, which has two internal restriction sites in the pSEAP-2 control DNA. Fig. 3a shows that ten out of seventy-five (13%) F0 generation piglets: 5 (not shown), 17, 26, 36, 40, 42, 43, 44, 48 and 64, had a 1.3 kb positive hybridization band. Animals 44–75 were obtained from a separate experiment (32 offspring from 8 gilts, total 30 gilts inseminated with a 28% pregnancy rate) to demonstrate reproducibility. Furthermore, analysis of sperm genomic DNA from a limited number of 8-month-old F0 generation animals (boars) showed that two out of eleven (18%), numbers 25 and 30, had a hybridization signal (data not shown).

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