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.
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.
Harel-Markowitz
Eliane
E
Koret School of Veterinary Medicine, Kimron Veterinary Institute, Bet Dagan, Israel.
Gurevich
Michael
M
Shore
Laurence S
LS
Katz
Adi
A
Stram
Yehuda
Y
Shemesh
Mordechai
M
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D002645:Chickens / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005260:Female; D043373:Follicle Stimulating Hormone, Human / Q000235:genetics* / Q000378:metabolism; D018628:Gene Dosage; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism; D014162:Transfection / Q000379:methods
D017931:DNA Primers; D043373:Follicle Stimulating Hormone, Human; D012333:RNA, Messenger; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins