Obtaining metaphase spreads from single blastomeres for PGD of chromosomal rearrangements.
It has previously been shown that it is possible to obtain metaphase chromosomes from single blastomeres converted into metaphase in the cytoplasm of a mouse zygote. This method is highly labour intensive and cannot be performed outside the preimplantation genetic diagnosis (PGD) laboratory, so to overcome these limitations, a method was developed for obtaining metaphase spreads from single biopsied blastomeres using different chemicals. The substances tested were calyculin A, caffeine, paclitaxel and colcemid in a total of 496 disaggregated and 234 biopsied blastomeres from day 3 embryos. It was demonstrated that the optimal method involved a combined use of 'selective biopsy' (selection of the biopsied blastomere according to morphological criteria) and exposure to caffeine. This resulted in shortening the mean incubation time of biopsied blastomeres, with a metaphase formation rate of 80%. The method is simple for obtaining metaphases from single blastomeres, and may be implemented in clinical practice of PGD for structural rearrangements.
Shkumatov
Artem
A
Reproductive Genetics Institute, 2825 North Halsted Street, Chicago, IL 60657, USA.
Kuznyetsov
Valeriy
V
Cieslak
Jeanine
J
Ilkevitch
Yuri
Y
Verlinsky
Yury
Y
D000818:Animals; D001757:Blastomeres / Q000378:metabolism*; D002110:Caffeine / Q000494:pharmacology; D002869:Chromosome Aberrations; D003470:Culture Media / Q000494:pharmacology*; D003593:Cytoplasm / Q000378:metabolism; D003703:Demecolcine / Q000494:pharmacology; D046149:Embryo Culture Techniques; D006801:Humans; D008387:Marine Toxins; D008677:Metaphase; D051379:Mice; D010080:Oxazoles / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D019836:Preimplantation Diagnosis; D015053:Zygote / Q000378:metabolism
D003470:Culture Media; D008387:Marine Toxins; D010080:Oxazoles; D002110:Caffeine; C059041:calyculin A; D017239:Paclitaxel; D003703:Demecolcine