Sexing and multiple genotype analysis from a single cell of bovine embryo.
We described a procedure for multiple genotype analysis (determination of sex and of three genetic markers) from a single cell derived from bovine preimplantation embryo. It consists of primer extension preamplification-polymerase chain reaction (PEP-PCR) and subsequent single assay or multiplex PCR. A single blastomere that was isolated by microaspiration from bovine embryos at the 16- to 32-cell stage then was lysed and was subjected to the PEP-PCR. When testing 75 embryos, efficiency of genotyping by standard PCR for kappa-casein, growth hormone (GH) and prolactin (PRL) polymorphic alleles was 91, 88 and 89%, respectively. Sexing efficiency in the multiplex PCR was 91%, based on the amplification of Y-specific locus using kappa-casein internal standard. The microaspiration of a single blastomere was shown not to be invasive for the embryos. It did not alter their development potential in vitro (P > 0.05), as was seen by obtaining a similar percentage of embryos developing further into the blastocyst stage in the group subjected to biopsy (44/75, 59%) and in the control group of embryos (30/50, 60%).
Chrenek
P
P
INRA, Biology of Development, Jouy-en-Josas, France. chrenekp@vuzv.sk
Boulanger
L
L
Heyman
Y
Y
Uhrin
P
P
Laurincik
J
J
Bulla
J
J
Renard
J P
JP
D000818:Animals; D001757:Blastomeres / Q000502:physiology*; D002364:Caseins / Q000032:analysis / Q000235:genetics; D002417:Cattle / Q000235:genetics* / Q000502:physiology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D005819:Genetic Markers; D005838:Genotype; D013006:Growth Hormone / Q000032:analysis / Q000235:genetics; D008297:Male; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D012150:Polymorphism, Restriction Fragment Length; D011388:Prolactin / Q000032:analysis / Q000235:genetics; D012732:Sex Determination Analysis / Q000379:methods / Q000662:veterinary*; D014998:Y Chromosome / Q000235:genetics
D002364:Caseins; D005819:Genetic Markers; D011388:Prolactin; D013006:Growth Hormone