Failure to produce transgenic offspring by intra-tubal insemination of gilts with DNA-treated sperm.
The reproducibility of the use of sperm cells as vectors of foreign DNA in the genome of pigs was verified in the present study and the effectiveness of four different procedures for sperm treatment was assessed. For each gilt, approximately 6 x 10(6) ejaculated boar spermatozoa were incubated for 30 min in 1 mL TALP medium containing 3 micrograms of linearized pSV2CAT plasmid DNA. Before incubation, spermatozoa were treated in four experimental groups: (1) cells were stored at 16 degrees C for 24 h and then washed three times in TALP; (2) cells from the fresh, undiluted sperm-rich fraction of an ejaculate were used immediately after collection, following the same procedure as (1); (3) cells were treated as in (2) with an extra wash; and (4) incubation with DNA was performed in TALP medium supplemented with 0.5 mg mL(-1) poly-L-lysine hydrobromide. As determined by immunolocalization, plasmid DNA molecules were found to be associated with 12-17.1% spermatozoa, depending on sperm treatment. Of 35 inseminated gilts, 20 gave birth to a total of 126 piglets. None of the piglets showed sign of exogenous DNA incorporation in any of the tissues tested, as assessed by the polymerase chain reaction and Southern blot. The potential of modifying the pig genome through "transformed' spermatozoa was not confirmed by these experiments.
Istituto di Anatomia degli Animali Domestici, Università degli Studi, Milano, Italy.
F
F
Gandolfi
M
M
Terqui
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S
Modina
T A
TA
Brevini
P
P
Ajmone-Marsan
F
F
Foulon-Gauzé
M
M
Courot
D000818:Animals; D030801:Animals, Genetically Modified; D015139:Blotting, Southern; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000032:analysis / Q000378:metabolism*; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D007315:Insemination, Artificial / Q000662:veterinary*; D008297:Male; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D013539:Simian virus 40 / Q000235:genetics; D013094:Spermatozoa / Q000378:metabolism*; D013552:Swine
D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase