Estimates of mRNA abundance in the mouse blastocyst based on cDNA library analysis

Studies of gene expression during blastocyst formation in mouse preimplantation development have been limited by the amount of RNA available per embryo. Our present approach to this problem has been to construct a large, representative, blastocyst cDNA library in λgtll. Random hexadeoxynucleotides were used as primers with total blastocyst RNA serving as template. RNA collected from 4,100 32–64 cell embryos was used to generate a library with an initial size of 30 × 106 recombinants. By using clone frequency as a measure of relative mRNA abundance, our data support previous work on the relative and absolute amounts of actin, histone H2a, and intracisternal A particle. Furthermore, we provide estimates for the abundance of cytokeratin endo A, cytokeratin endo B, and β‐tubulin from clone frequency data. Insert sizes for isolated clones range from 200 bp to 3.6 kb with full‐length or near‐full‐length insert sizes for selected clones, indicating that random primer methods generate cDNAs which can represent a significant portion of the mRNA. We have so far characterized products whose abundance is equal to or greater than 0.002% of total RNA. This library offers the potential for the analyses of presumptive regulatory gene products in the mouse preimplantation embryo which are represented as low abundance (<1% of mRNA) RNAs.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  P. Newman,et al.  Enzymatic amplification of specific cDNA inserts from λgt11 libraries , 1988 .

[3]  T. Yen,et al.  Autoregulated instability of β-tubulin mRNAs by recognition of the nascent amino terminus of βtubulin , 1988, Nature.

[4]  L. Pikó,et al.  Patterns of mRNA prevalence and expression of B1 and B2 transcripts in early mouse embryos. , 1987, Development.

[5]  J. C. Chisholm,et al.  Cytokeratin filament assembly in the preimplantation mouse embryo. , 1987, Development.

[6]  M. Haussler,et al.  Molecular cloning of complementary DNA encoding the avian receptor for vitamin D. , 1987, Science.

[7]  J. Herz,et al.  Efficient construction of cDNA libraries in plasmid expression vectors using an adaptor strategy. , 1986, Nucleic acids research.

[8]  C. Watson,et al.  Construction of a representative cDNA library from mRNA isolated from mouse oocytes , 1986, FEBS letters.

[9]  R. Oshima,et al.  Molecular cloning and characterization of the Endo B cytokeratin expressed in preimplantation mouse embryos. , 1986, The Journal of biological chemistry.

[10]  G. Kidder,et al.  Timing of transcription and protein synthesis underlying morphogenesis in preimplantation mouse embryos. , 1985, Developmental biology.

[11]  D. L. Weeks,et al.  Identification and cloning of localized maternal RNAs from xenopus eggs , 1985, Cell.

[12]  G. Kidder,et al.  Utilization of cytoplasmic poly(A)+RNA for protein synthesis in preimplantation mouse embryos. , 1985, Journal of embryology and experimental morphology.

[13]  G. Schultz,et al.  Quantitative and qualitative changes in histone gene expression during early mouse embryo development. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[14]  R. Graves,et al.  Differential expression of two clusters of mouse histone genes. , 1985, Journal of molecular biology.

[15]  P. Brûlet,et al.  One gene and one pseudogene for the cytokeratin endo A. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[16]  G. Schultz,et al.  Actin mRNA content in normal and delayed implanting mouse embryos. , 1985, Developmental biology.

[17]  B. Hoffman,et al.  A simple and very efficient method for generating cDNA libraries. , 1983, Gene.

[18]  I. Dawid,et al.  Differential gene expression in the gastrula of Xenopus laevis. , 1983, Science.

[19]  A. Feinberg,et al.  A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. , 1983, Analytical biochemistry.

[20]  D. Bogenhagen,et al.  Clusters of point mutations are found exclusively around rearranged antibody variable genes. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[21]  L. Pikó,et al.  Quantitative changes in total RNA, total poly(A), and ribosomes in early mouse embryos. , 1982, Developmental biology.

[22]  M. Sherman,et al.  Effects of α-amanitin on programming of mouse blastocyst development , 1981 .

[23]  F. Gros,et al.  Mouse actin messenger RNAs. Construction and characterization of a recombinant plasmid molecule containing a complementary DNA transcript of mouse alpha-actin mRNA. , 1981, The Journal of biological chemistry.

[24]  M. Cole,et al.  Sequence organization of cloned intracisternal a particle genes , 1980, Cell.

[25]  N. Davidson,et al.  The actin genes of drosophila: a dispersed multigene family , 1980, Cell.

[26]  P. Braude.  Time-dependent effects of α-amanitin on blastocyst formation in the mouse , 1979 .

[27]  H. Pelham,et al.  A microsystem for the extraction and in-vitro translation of mouse embryo mRNA. , 1979, Journal of reproduction and fertility.

[28]  R. Brinster,et al.  Synthesis of tubulin and actin during the preimplantation development of the mouse. , 1978, Experimental cell research.

[29]  A. Handyside,et al.  Temporal and spatial patterns of the synthesis of tissue-specific polypeptides in the preimplantation mouse embryo. , 1978, Journal of embryology and experimental morphology.

[30]  W. D. Benton,et al.  Screening lambdagt recombinant clones by hybridization to single plaques in situ. , 1977, Science.

[31]  M. Nemer.  Developmental changes in the synthesis of sea urchin embryo messenger RNA containing and lacking polyadenylic acid , 1975, Cell.

[32]  C. Milcarek,et al.  The metabolism of a poly(A) minus mRNA fraction in HeLa cells. , 1974, Cell.

[33]  J. Darnell,et al.  Biogenesis and characterization of histone messenger RNA in HeLa cells. , 1972, Journal of molecular biology.

[34]  L. Pikó,et al.  Amounts, synthesis, and some properties of intracisternal A particle-related RNA in early mouse embryos. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[35]  R. Gardner.  The relationship between cell lineage and differentiation in the early mouse embryo. , 1978, Results and problems in cell differentiation.