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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

Expression of secreted human alkaline phosphatase (SEAP) from the transgenic pigs. Sera from 70-day-old piglets from nontransgenic controls and group I F0 generation animals were analyzed by using Great Escape SEAP Chemiluminescence Detection kit (Clontech Laboratories Inc). The average reading for controls is 6.9 × 103 ± 3.7 × 103 RLU. (●) represents nontransgenic nonheat-treated sera. (▲) represents transgenic nonheat-treated sera. (■) represents nontransgenic heat-treated sera. (▼) represents transgenic heat-treated sera. The breed, number, and sex of nontransgenic controls are: Duroc, 144(F), 145(F), 146(M), 153(M), 161(F), 162(M) and 163(M); Yorkshire, 141(M), 142(M), 143(M), 150(M), 151(F) and 152(F); Landrace, 138(M), 139(M), 140(M), 147(M), 148(M), 149(M), 156(F), 157(F), 158(F), 159(F) and 160(F).
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Figure 4: Expression of secreted human alkaline phosphatase (SEAP) from the transgenic pigs. Sera from 70-day-old piglets from nontransgenic controls and group I F0 generation animals were analyzed by using Great Escape SEAP Chemiluminescence Detection kit (Clontech Laboratories Inc). The average reading for controls is 6.9 × 103 ± 3.7 × 103 RLU. (●) represents nontransgenic nonheat-treated sera. (▲) represents transgenic nonheat-treated sera. (■) represents nontransgenic heat-treated sera. (▼) represents transgenic heat-treated sera. The breed, number, and sex of nontransgenic controls are: Duroc, 144(F), 145(F), 146(M), 153(M), 161(F), 162(M) and 163(M); Yorkshire, 141(M), 142(M), 143(M), 150(M), 151(F) and 152(F); Landrace, 138(M), 139(M), 140(M), 147(M), 148(M), 149(M), 156(F), 157(F), 158(F), 159(F) and 160(F).

Mentions: As an alternative method to detect transgenic pigs, we have also looked for the expression of heat stable SEAP in the sera collected from the 70-day-old F0 generation animals. The results are summarized in Fig. 4. In the control study, the chemiluminescent signals range from 4 × 106 to 2 × 107 RLU when assayed without the heat deactivation step as displayed in the top panel of Fig. 4. There was no obvious difference in the level of total alkaline phosphatase activity between the genetically modified F0 generation animals and nontransgenic controls, nor was any difference observed in the controls of the different breeds. The bottom panel of Fig. 4 shows the results of an assay after heat inactivation of the endogenous alkaline phosphatase activity. A reading of more than 2 × 104 RLU of SEAP activity was set as positive in the genetically modified pigs, which is significantly higher than in the nontransgenic controls (6.9 × 103 ± 3.7 × 103 RLU). Thus, twenty-one out of thirty-six (58%) group one piglets (F0 generation) were shown to express the thermostable human SEAP enzyme. The sera from the second group of F0 generation animals (#44–75) were assayed and showed similar results: fourteen out of twenty-two (64%) pigs showed comparable levels of expressed SEAP (>2 × 104 RLU) (data not shown). In both experiments, due to the mosaic condition in many of the transgenic pigs, the number of F0 generation animals expressing human SEAP in their sera (average 60%), which might be secreted from different organs or cells, is greater than the number of animals found to be positive (13%) by Southern blot analysis of a small localized area (tail tip).


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)

Expression of secreted human alkaline phosphatase (SEAP) from the transgenic pigs. Sera from 70-day-old piglets from nontransgenic controls and group I F0 generation animals were analyzed by using Great Escape SEAP Chemiluminescence Detection kit (Clontech Laboratories Inc). The average reading for controls is 6.9 × 103 ± 3.7 × 103 RLU. (●) represents nontransgenic nonheat-treated sera. (▲) represents transgenic nonheat-treated sera. (■) represents nontransgenic heat-treated sera. (▼) represents transgenic heat-treated sera. The breed, number, and sex of nontransgenic controls are: Duroc, 144(F), 145(F), 146(M), 153(M), 161(F), 162(M) and 163(M); Yorkshire, 141(M), 142(M), 143(M), 150(M), 151(F) and 152(F); Landrace, 138(M), 139(M), 140(M), 147(M), 148(M), 149(M), 156(F), 157(F), 158(F), 159(F) and 160(F).
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Related In: Results  -  Collection

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Figure 4: Expression of secreted human alkaline phosphatase (SEAP) from the transgenic pigs. Sera from 70-day-old piglets from nontransgenic controls and group I F0 generation animals were analyzed by using Great Escape SEAP Chemiluminescence Detection kit (Clontech Laboratories Inc). The average reading for controls is 6.9 × 103 ± 3.7 × 103 RLU. (●) represents nontransgenic nonheat-treated sera. (▲) represents transgenic nonheat-treated sera. (■) represents nontransgenic heat-treated sera. (▼) represents transgenic heat-treated sera. The breed, number, and sex of nontransgenic controls are: Duroc, 144(F), 145(F), 146(M), 153(M), 161(F), 162(M) and 163(M); Yorkshire, 141(M), 142(M), 143(M), 150(M), 151(F) and 152(F); Landrace, 138(M), 139(M), 140(M), 147(M), 148(M), 149(M), 156(F), 157(F), 158(F), 159(F) and 160(F).
Mentions: As an alternative method to detect transgenic pigs, we have also looked for the expression of heat stable SEAP in the sera collected from the 70-day-old F0 generation animals. The results are summarized in Fig. 4. In the control study, the chemiluminescent signals range from 4 × 106 to 2 × 107 RLU when assayed without the heat deactivation step as displayed in the top panel of Fig. 4. There was no obvious difference in the level of total alkaline phosphatase activity between the genetically modified F0 generation animals and nontransgenic controls, nor was any difference observed in the controls of the different breeds. The bottom panel of Fig. 4 shows the results of an assay after heat inactivation of the endogenous alkaline phosphatase activity. A reading of more than 2 × 104 RLU of SEAP activity was set as positive in the genetically modified pigs, which is significantly higher than in the nontransgenic controls (6.9 × 103 ± 3.7 × 103 RLU). Thus, twenty-one out of thirty-six (58%) group one piglets (F0 generation) were shown to express the thermostable human SEAP enzyme. The sera from the second group of F0 generation animals (#44–75) were assayed and showed similar results: fourteen out of twenty-two (64%) pigs showed comparable levels of expressed SEAP (>2 × 104 RLU) (data not shown). In both experiments, due to the mosaic condition in many of the transgenic pigs, the number of F0 generation animals expressing human SEAP in their sera (average 60%), which might be secreted from different organs or cells, is greater than the number of animals found to be positive (13%) by Southern blot analysis of a small localized area (tail tip).

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