TY - JOUR
T1 - Use of sperm plasmid DNA lipofection combined with REMI (Restriction Enzyme-mediated Insertion) for production of transgenic chickens expressing eGFP (Enhanced Green Fluorescent Protein) or human follicle-stimulating hormone
AU - Harel-Markowitz, Eliane
AU - Gurevich, Michael
AU - Shore, Laurence S.
AU - Katz, Adi
AU - Stram, Yehuda
AU - Shemesh, Mordechai
PY - 2009/5
Y1 - 2009/5
N2 - Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.
AB - Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.
KW - Follicle-stimulating hormone
KW - Lipofection
KW - Restriction enzyme
KW - Sperm
KW - Transgenic chickens
UR - http://www.scopus.com/inward/record.url?scp=65949089365&partnerID=8YFLogxK
U2 - 10.1095/biolreprod.108.070375
DO - 10.1095/biolreprod.108.070375
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C2 - 19164177
AN - SCOPUS:65949089365
SN - 0006-3363
VL - 80
SP - 1046
EP - 1052
JO - Biology of Reproduction
JF - Biology of Reproduction
IS - 5
ER -