Construction of primary and subtracted cDNA libraries from early embryos.
By modifying current cDNA cloning and electroporation methods, large and representative murine cDNA libraries were synthesized from 10 to 100 ng mRNA isolated from unfertilized egg and preimplantation mouse embryos. High cloning efficiency is essential for complete representation of genes expressed in egg and preimplantation embryos and for the isolation of stage-specific genes using subtractive hybridization. Because the mouse embryo contains no more than 50 pg of poly(A)+ mRNA at any stage of preimplantation development, approximately 5000-10,000 embryos are required to obtain enough mRNA to synthesize libraries using current methods. To obtain a representative library that also includes rare transcripts, the size of the library should be at least 10(6) clones. The average percent conversion of mRNA to single-stranded cDNA was 20-40%, so that a cloning efficiency of nearly 2 x 10(8) cfu/microgram cDNA is required for such a cDNA library. No previous methods have provided directional cloning of cDNA into plasmids with these high efficiencies. The advent of electroporation methods for the introduction of nucleic acids into bacteria has made possible the use of standard plasmid vectors for high-efficiency cDNA cloning. Plasmid vectors are currently available that can accommodate the directional cloning of cDNA such that T7 and T3 RNA polymerase promoter sequences can be used to generate sense and anti-sense transcripts for subtractive hybridization and riboprobe synthesis. The cDNA libraries we derived using this methodology are a reusable and abundant source of genetic information about the control of preimplantation development. Specialized subtractive cDNA libraries enriched for genes expressed exclusively at a predetermined time in development give access to genes expressed in a stage-specific manner. The ability to construct new cDNA libraries from limited amounts of starting material ensures the provision of new and important resources for the identification and study of novel genes or gene families, and it is an important new tool for understanding the molecular control of mammalian development.
Rothstein
J L
JL
Department of Microbiology/Immunology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Johnson
D
D
Jessee
J
J
Skowronski
J
J
DeLoia
J A
JA
Solter
D
D
Knowles
B B
BB
D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis / Q000096:biosynthesis*; D018274:Electroporation / Q000379:methods; D004622:Embryo, Mammalian / Q000378:metabolism*; D015723:Gene Library; D008322:Mammals; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011061:Poly A / Q000032:analysis / Q000096:biosynthesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis*; D015183:Restriction Mapping; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA