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PCR-generated cDNA libraries from reduced numbers of mouse oocytes

  • Frédérique Revel (a1), Jean-Paul Renard (a1) and Véronique Duranthon (a1)


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.


Corresponding author

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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Bachvarova, R. (1988). Small B2 RNAs in mouse oocytes, embryos, and somatic tissues. Dev. Biol. 130 513523.
Bachvarova, R., De Leon, V., Johnson, A., Kaplan, G. & Paynton, B.V. (1985). Changes in total RNA, polyadenylated RNA, and actin mRNA during meiotic maturation of mouse oocytes. Dev. Biol. 108, 325331.
Beliavsky, A., Vinogradova, T. & Rajewsky, K. (1989). PCR based cDNA library construction: general cDNA libraries at the level of a few cells. Nucleic Acids Res. 17, 2919–32.
Brady, G.Barbara, M.& Iscove, N. (1990). Representative in vitro cDNA amplification from individual hematopoietic cells and colonies. Methods Mol. Cell. Biol. 2, 1725.
Brunet, J.F.Shapiro, E.Foster, S.A.Kandel, E.R.& Iino, Y. (1991). Identification of a peptide specific for Aplysia sensory neurons by PCR-based differential screening. Science. 252, 856–9.
Church, G.M.& Gilbert, W. (1984). Genomic sequencing.Proc. Natl. Acad. Sci. USA. 81, 1991–5.
Clegg, K.B.& Piko, L. (1983). Quantitative aspects of RNA synthesis and polyadenylation in 1-cell and 2-cell mouse embryos. J. Embryol. Exp. Morphol. 74, 169–82.
Copp, A. (1995). Death before birth: clues from gene knockouts and mutations. Trends Genet. 11, 8793.
Dumas, B. (1980). Construction de plasmides en vue de l'étude de la notion de promoteur chez les eucaryotes. Diplome d'éetude approfondie de biochimie fondamentale, University of Paris VII.
Feinberg, A.P.& Vogelstein, B. (1984). A technique for radio-labeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137, 266–7.
Fregien, N.Dolecki, G.J.Mandel, M.& Humphreys, T. (1993). Molecular cloning of live individual stage and tissue-specific mRNA sequences from sea urchin pluteus embryos. Mot. Cell. Biol. 3, 1021–31.
Goldman, D.S., Kiessling, A.A., & Cooper, G.M. (1988). Post-transcriptional processing suggest that c-mos functions as a maternal message in mouse eggs. Oncogene 3, 159–62.
Keshet, E.Rosenberg, M.P.Mercer, J.A.Propst, F.Vande Woude, G.F.Jenkins, N.A.& Copeland, N.G. (1988). Developmental regulation of ovarian-specific mos expression. Oncogene 2, 235–40.
Lalanne, J.L.Bregegere, F.Delarbre, C.Abaatado, J.P.Gachelin, C.& Kourilsky, p. (1982). Comparison of nucleotide sequences of mRNAs belonging to the mouse H-2 multigene family. Nucleic Acid Res. 10, 1039–48.
Latham, K.E.Garrels, J.L.Chang, C.& Solter, D. (1991). Quantitative analysis of protein synthesis in mouse embryos. I. Extensive reprogramming of the one- and two-cell stages. Development 112, 921–32.
Latham, K.E.Garel, J.I.& Solter, D. (1994). Alterations in protein synthesis following transplantation of mouse 8-cell stage nuclei to enucleated 1-cell embryos. Dev. Biol. 163, 341–50.
Luqmani, Y.A.& Lymboura, M. (1994).Subtraction hybridization cloning of RNA amplified from different cell population microdissected from cryostat tissue section. Anal. Biochem. 222, 102–9.
Maniatis, T.Fritsch, E.F.& Sambrook, J. (1982). Molecular Cloning: A laboratory Manual. New York: Cold Spring Harbor Laboratory.
McConnel, J.& Watson, C.J. (1986). Construction of a representative cDNA library from mRNA isolated from mouse oocytes. FEBS Lett. 195, 99203.
Miyamoto, H.Matsushiro, A.& Nozaki, M. (1993). Molecular cloning of a novel mRNA sequence expressed in cleavage stage mouse embryos. Mol. Reprod. Dev. 34 17.
Moreau, J.Touzalen, N. & Mechali, M. (1995). Isolation of cDNAs from maternal mRNAs specifically present during early development. Mol. Reprod. Dev. (in press).
Nozaki, M.Murata, K.Morita, T.& Matsushiro, A. (1988). Isolation of ENDO A cDNA from mouse 8-cell stage embryos. Biochem. Biophys. Res. Commun. 154, 890–4.
Paris, J.& Philippe, M. (1990). Poly(A) metabolism and polysomal recruitment of maternal mRNAs during early Xenopus development. Dev. Biol. 140, 221–4
Piko, L.& Clegg, K.B. (1982). Quantitative changes in total RNA, total poly(A) and ribosomes in early mouse embryos. Dev. Biol. 89, 362–78.
Poirier, F.Chan, C.T.J.Timmons, P.M.Robertson, E.J.Evans, M.J.& Rigby, P.W. (1991). The murine H19 gene is activated during embryonic stem cell differentiation in vitro and at the time of implantation in the developing embryo. Development. 113, 1105–4.
Richoux-Duranthon, V. (1991). Rôle des effets parentaux dana le developpement de l'embryon. Etude à partir d'un système modèle: Is lignèe de souris DDK. Thesis, University of Paris VI.
Rothstein, J.L.Johnson, D.Deloia, J.A.Skowronski, J.& Solter, D. (1992). Gene expression during preimplantation mouse development. Genes Dev. 6, 1190–201.
Sargent, T.D.& Dawid, I.G. (1983). Differential gene expression in the gastrula of Xenopus laevis. Science 222, 135–9.
Smith, D.& Gridley, T. (1992). Differential screening of a PCR-generated mouse embryo cDNA library: glucose transporters are differentially expressed in early postimplantation mouse embryos. Development. 161, 555–61.
Swalla, B.J.Makabe, K.W.Satoh, N.& Jeffery, R.W. (1993). Novel genes expressed differentially in ascidians with alternate modes of development. Development. 119, 307–18.
Taylor, K.D.& Piko, L. (1987). Pattern of mRNA prevalence and expression of B1 and B2 transcripts in early mouse embryos. Development. 101, 877–92.
Temeles, G.L.Ram, P.T.Rothstein, J.L.& Schultz, R.M. (1994). Expression patterns of novel genes during mouse preimplantation embryogenesis. Mol. Reprod. Dev. 37, 121–9.
Thompson, E.M.Christians, E.Stinnakre, M.G.& Renard, J. P. (1994). Scaffold-attachment regions stimulate HSP70.1 expression in mouse preimplantation embryos but not in differentiated tissues. Mol. Cell. Biol. 14, 4694–703.
Urven, L.E.Weng, D.E.Schumaker, A.L.Gearhart, J.D.& McCarrey, J.R. (1993). Differential gene expression in fetal mouse germ cells. Biol. Reprod. 48, 564–74.
Varmuza, S.& Tate, p. (1992). Isolation of epiblast-specific cDNA clones by differential hybridization with polymerase chain reaction-amplified probes derived from single embryos. Mol. Reprod. Dev. 32, 339–48.
Welsh, J.Liu, J.P.& Efstratiadis, A. (1990). Cloning of PCR-amplified total cDNA: construction of a mouse oocyte cDNA library. Genet. Anal. Techn. Appl. 7, 517.
Weng, D.E.Morgan, R.A.& Gearhart, J.D. (1989). Estimates of mRNA abundance in the mouse blastocyst based on cDNA library analysis. Mol. Reprod. Dev. 1, 233–41.


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PCR-generated cDNA libraries from reduced numbers of mouse oocytes

  • Frédérique Revel (a1), Jean-Paul Renard (a1) and Véronique Duranthon (a1)


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