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PCR-generated cDNA libraries from reduced numbers of mouse oocytes

Published online by Cambridge University Press:  26 September 2008

Frédérique Revel ,
Jean-Paul Renard  and
Véronique Duranthon
Frédérique Revel*
Affiliation:
Unité de biologie du développement INRA, Jouy en Josas, France
Jean-Paul Renard
Affiliation:
Unité de biologie du développement INRA, Jouy en Josas, France
Véronique Duranthon
Affiliation:
Unité de biologie du développement INRA, Jouy en Josas, France
*
F. Revel, Unité de biologie du développement, INRA, F78352 Jouy en Josas, France. Telephone: (33) 1 34652592. (33) 1 34652877.0.
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Summary

We describe a rapid and reproducible method for cloning cDNA amplified from 10 mouse oocytes. The procedure consists in priming cDNA synthesis from a crude cellular extract using an oligo d(T) containing primer and submitting the size-limited cDNA first strand to poly(dG) tailing. The whole cDNA population is then polymerase chain reaction (PCR) amplified using two primers complementary to oligo d(A) and oligo d(G) ends of the cDNA. In this procedure no purification steps are required. We obtained about 5 ×106 clones from 10 oocytes. Screening of the library showed that the relative abundance of the transcripts was preserved during amplification and cloning and that the procedure allows cloning of low-abundance sequences at least as rare as 0.008% of the mRNA. The repeatable generation of representative cDNA libraries from reduced numbers of oocytes or embryos should open new opportunities for obtaining genetic information from mammalian preimplantation embryos.

Keywords

DNA libraryMammalian oocytespolymerase chain reactionPreimplantation embryos
Type
Article
Information
Zygote , Volume 3 , Issue 3 , August 1995 , pp. 241 - 250
Copyright
Copyright © Cambridge University Press 1995

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