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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

FISH analysis for transgene localization. The transgene was located to chromosome 15, region q25–q28, in TG pig #152 (F1 generation) by FISH. A 3.1 kb EcoR I/Sal I DNA fragment from the plasmid vector region of the pSEAP2-Control (# 2308–260) was used as a probe. The standard FISH experiment was performed by SeeDNA Biotech Inc. (Windsor, Ontario, Canada). The detailed localization was further determined based on the summary from ten photos. DAPI (4', 6'-Diamidino-2-phenylindole Dihydrochloride) is a DNA-specific fluorescent dye. FITC: fluorescein isothiocyanate.
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Figure 5: FISH analysis for transgene localization. The transgene was located to chromosome 15, region q25–q28, in TG pig #152 (F1 generation) by FISH. A 3.1 kb EcoR I/Sal I DNA fragment from the plasmid vector region of the pSEAP2-Control (# 2308–260) was used as a probe. The standard FISH experiment was performed by SeeDNA Biotech Inc. (Windsor, Ontario, Canada). The detailed localization was further determined based on the summary from ten photos. DAPI (4', 6'-Diamidino-2-phenylindole Dihydrochloride) is a DNA-specific fluorescent dye. FITC: fluorescein isothiocyanate.

Mentions: To further demonstrate that the transgene was permanently integrated into pig genomic DNA, a FISH (fluorescence in situ hybridization) experiment was performed using a blood sample from a transgenic F1 pig (#152). As shown in Fig. 5, the transgene was located to chromosome 15, region q25–q28. Moreover, two out of nine progeny (F2) of this TG pig showed positive transgene hybridization bands by Southern blot analysis (Fig. 6). Therefore, we have demonstrated that the transgene can be stably transmitted to future generations in this line of pigs.


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)

FISH analysis for transgene localization. The transgene was located to chromosome 15, region q25–q28, in TG pig #152 (F1 generation) by FISH. A 3.1 kb EcoR I/Sal I DNA fragment from the plasmid vector region of the pSEAP2-Control (# 2308–260) was used as a probe. The standard FISH experiment was performed by SeeDNA Biotech Inc. (Windsor, Ontario, Canada). The detailed localization was further determined based on the summary from ten photos. DAPI (4', 6'-Diamidino-2-phenylindole Dihydrochloride) is a DNA-specific fluorescent dye. FITC: fluorescein isothiocyanate.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: FISH analysis for transgene localization. The transgene was located to chromosome 15, region q25–q28, in TG pig #152 (F1 generation) by FISH. A 3.1 kb EcoR I/Sal I DNA fragment from the plasmid vector region of the pSEAP2-Control (# 2308–260) was used as a probe. The standard FISH experiment was performed by SeeDNA Biotech Inc. (Windsor, Ontario, Canada). The detailed localization was further determined based on the summary from ten photos. DAPI (4', 6'-Diamidino-2-phenylindole Dihydrochloride) is a DNA-specific fluorescent dye. FITC: fluorescein isothiocyanate.
Mentions: To further demonstrate that the transgene was permanently integrated into pig genomic DNA, a FISH (fluorescence in situ hybridization) experiment was performed using a blood sample from a transgenic F1 pig (#152). As shown in Fig. 5, the transgene was located to chromosome 15, region q25–q28. Moreover, two out of nine progeny (F2) of this TG pig showed positive transgene hybridization bands by Southern blot analysis (Fig. 6). Therefore, we have demonstrated that the transgene can be stably transmitted to future generations in this line of pigs.

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