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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

Ionic interaction analysis of mAb C and DNA. 0.3 μg of Sal I-linearized pSEAP-2 control DNA was mixed with water, medium or mAb C for 40 minutes at room temperature with occasional mixing and then subjected to 1% agarose gel electrophoresis. Lane 1: DNA in dH2O; lane 2 and 8: DNA in Modified Tyrode's medium without BSA (MTM); lane 3: DNA plus 0.1 μg mAb C in MTM; lane 4: DNA plus 0.3 μg mAb C in MTM; lane 5: DNA plus 1 μg mAb C in MTM; lane 6: DNA plus 3 μg mAb C in MTM; and lane 7: DNA plus 10 μg mAb C in MTM.
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Figure 2: Ionic interaction analysis of mAb C and DNA. 0.3 μg of Sal I-linearized pSEAP-2 control DNA was mixed with water, medium or mAb C for 40 minutes at room temperature with occasional mixing and then subjected to 1% agarose gel electrophoresis. Lane 1: DNA in dH2O; lane 2 and 8: DNA in Modified Tyrode's medium without BSA (MTM); lane 3: DNA plus 0.1 μg mAb C in MTM; lane 4: DNA plus 0.3 μg mAb C in MTM; lane 5: DNA plus 1 μg mAb C in MTM; lane 6: DNA plus 3 μg mAb C in MTM; and lane 7: DNA plus 10 μg mAb C in MTM.

Mentions: mAb C is a positively charged, basic protein and all DNA molecules are negatively charged. To study mAb C's capability to interact with DNA, we added an increasing amount of mAb C to a constant amount of Sal I-linearized pSEAP-2 control DNA encoding for secreted human alkaline phosphatase under the control of the SV40 early promoter and the SV40 enhancer. This resulted in its partial neutralization and the formation of an antibody-DNA complex with various electrical charges as can be seen when the mixtures are separated by agarose gel electrophoresis (Fig. 2). DNA smears caused by diverse rates of migration in the gel when compared with the controls can be observed in lanes 4 and 5. When the amount of added mAb C reached 3 μg or more in lane 6 and 7, the resulting complexes had a net electrical charge of zero and no longer responded to the electric field in the gel, remaining in the original sample loading point. This suggests that the DNA interacts with mAb C via ionic interaction.


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)

Ionic interaction analysis of mAb C and DNA. 0.3 μg of Sal I-linearized pSEAP-2 control DNA was mixed with water, medium or mAb C for 40 minutes at room temperature with occasional mixing and then subjected to 1% agarose gel electrophoresis. Lane 1: DNA in dH2O; lane 2 and 8: DNA in Modified Tyrode's medium without BSA (MTM); lane 3: DNA plus 0.1 μg mAb C in MTM; lane 4: DNA plus 0.3 μg mAb C in MTM; lane 5: DNA plus 1 μg mAb C in MTM; lane 6: DNA plus 3 μg mAb C in MTM; and lane 7: DNA plus 10 μg mAb C in MTM.
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Related In: Results  -  Collection

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Figure 2: Ionic interaction analysis of mAb C and DNA. 0.3 μg of Sal I-linearized pSEAP-2 control DNA was mixed with water, medium or mAb C for 40 minutes at room temperature with occasional mixing and then subjected to 1% agarose gel electrophoresis. Lane 1: DNA in dH2O; lane 2 and 8: DNA in Modified Tyrode's medium without BSA (MTM); lane 3: DNA plus 0.1 μg mAb C in MTM; lane 4: DNA plus 0.3 μg mAb C in MTM; lane 5: DNA plus 1 μg mAb C in MTM; lane 6: DNA plus 3 μg mAb C in MTM; and lane 7: DNA plus 10 μg mAb C in MTM.
Mentions: mAb C is a positively charged, basic protein and all DNA molecules are negatively charged. To study mAb C's capability to interact with DNA, we added an increasing amount of mAb C to a constant amount of Sal I-linearized pSEAP-2 control DNA encoding for secreted human alkaline phosphatase under the control of the SV40 early promoter and the SV40 enhancer. This resulted in its partial neutralization and the formation of an antibody-DNA complex with various electrical charges as can be seen when the mixtures are separated by agarose gel electrophoresis (Fig. 2). DNA smears caused by diverse rates of migration in the gel when compared with the controls can be observed in lanes 4 and 5. When the amount of added mAb C reached 3 μg or more in lane 6 and 7, the resulting complexes had a net electrical charge of zero and no longer responded to the electric field in the gel, remaining in the original sample loading point. This suggests that the DNA interacts with mAb C via ionic interaction.

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