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Transgene transmission in chickens by sperm-mediated gene transfer after seminal plasma removal and exogenous DNA treated with dimethylsulfoxide or N,N-dimethylacetamide

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Abstract

Transgenic animals have been successfully produced by mass gene transfer techniques such as sperm-mediated gene transfer (SMGT). The aim of this work was to demonstrate transgene transmission by SMGT in chickens using dimethylsulfoxide (DMSO) or N,N-dimethylacetamide (DMAc) as transfectants after seminal plasma removal to prevent DNase activity. Sperm samples were prepared by repetitive washes, and after each wash sperm motility, seminal plasma proteins, exogenous DNA integrity and its uptake by spermatozoa were evaluated. Laying hens were inseminated using spermatozoa transfected with pEGFP-N1 vector in the presence of DMSO or DMAc. Transgene transmission in newborn chicks was evaluated by in vivo enhanced green fluorescent protein (EGFP) expression, RT-PCR and PCR analysis. DNA internalization was limited to sperm samples washed twice. The presence of DMSO or DMAc during transfection had no effect on fertilization or hatching rates. PCR analysis detected the presence of EGFP DNA in 38% of newborn chicks from the DMSO group and 19% from the DMAc group. EGFP mRNA was detected in 21% of newborn chicks from the DMSO group, as against 8.5% from the DMAc group. However, in vivo expression of EGFP was only observed in a single animal from the DMSO group. Our data revealed that the plasmid DNA–DMSO combination coupled with sperm washes can be an efficient method for transfection in chickens.

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Abbreviations

CMV:

cytomegalovirus

DMAc:

N,N-dimethylacetamide

DMSO:

dimethylsulfoxide

EGFP:

enhanced green fluorescent protein

PBS:

phosphate buffered saline

REMI:

restriction-mediated insertion

SMGT:

sperm-mediated gene transfer

TMGT:

testis-mediated gene transfer

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Acknowledgements

The authors thank Dr Alan McBride for his critical review of the manuscript. This work was supported by CNPq and FAPERGS. VFC and PMML are students of the Graduate Program in Biotechnology at Universidade Federal de Pelotas and are supported by CAPES. JCD and OAD are research fellows of CNPq.

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Authors and Affiliations

  1. Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Campus Universitário, Caixa Postal 354, CEP 96010-900, Pelotas, RS, Brazil

    Tiago Collares, Vinicius Farias Campos, Priscila Marques Moura de Leon, Marta G Amaral, Odir A Dellagostin, João Carlos Deschamps & Fabiana K Seixas

  2. Programa de Pós-Graduação em Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av Prof Dr Orlando Marques de Paiva, 87, CEP 05508 270, Cidade Universitária, São Paulo, SP, Brazil

    Paulo V Cavalcanti

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  1. Tiago Collares
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  2. Vinicius Farias Campos
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  3. Priscila Marques Moura de Leon
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  4. Paulo V Cavalcanti
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  5. Marta G Amaral
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  6. Odir A Dellagostin
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  7. João Carlos Deschamps
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  8. Fabiana K Seixas
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Corresponding author

Correspondence to Tiago Collares.

Additional information

Corresponding editor: Seyed E Hasnain

[Collares T, Campos VF, de Leon PMM, Cavalcanti PV, Amaral MG, Dellagostin OA, Deschamps JC and Seixas FK 2011 Transgene transmission in chickens by sperm-mediated gene transfer after seminal plasma removal and exogenous DNA treated with dimethylsulfoxide or N,N-dimethylacetamide. J. Biosci. 36 613–620] DOI 10.1007/s12038-011-9098-x

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Collares, T., Campos, V.F., de Leon, P.M.M. et al. Transgene transmission in chickens by sperm-mediated gene transfer after seminal plasma removal and exogenous DNA treated with dimethylsulfoxide or N,N-dimethylacetamide. J Biosci 36, 613–620 (2011). https://doi.org/10.1007/s12038-011-9098-x

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  • DOI: https://doi.org/10.1007/s12038-011-9098-x

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