Construction of a mouse blastocyst cDNA library by PCR amplification from total RNA.
Studies of the development and differentiation of early mammalian embryos have been severely limited by the paucity of material. Such studies have been largely restricted to the examination of abundant genes/proteins or to developmental expression studies of known genes for which DNA sequence data are available, allowing the use of reverse transcription and polymerase chain reaction amplification (RT-PCR). To eliminate the need for hundreds or thousands of oocytes or embryos in the construction of representative cDNA libraries, we describe a technique for generating and cloning cDNA using small caesium chloride gradient centrifugation to isolate total RNA from oocytes or embryos, followed by RT-PCR of mRNA from this total RNA. Total RNA was isolated from 70 mouse blastocysts. A portion of the cDNA generated (equivalent to seven blastocysts) was cloned, yielding a mouse blastocyst cDNA library of 1 million clones. We show that the library is representative in that it contains beta-actin, intracisternal A-type particles, tissue plasminogen activator, and B1 and B2 repetitive elements in frequencies comparable with published data from conventionally constructed libraries and estimates of mRNA abundance from expression studies. Furthermore, DNA sequencing of 22 clones chosen at random and compared with DNA sequence databases shows that approximately half are novel sequences. These data demonstrate that representative cDNA libraries can be constructed in situations where cell numbers are limiting and will facilitate the isolation of novel and interesting clones.
Corrick
C M
CM
Molecular Genetics and Development Group, Monash University, Clayton, Victoria, Australia.
Silvestro
M J
MJ
Lahoud
M H
MH
Allen
G J
GJ
Tymms
M J
MJ
Kola
I
I
D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000502:physiology*; D002454:Cell Differentiation; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D015342:DNA Probes; D018076:DNA, Complementary; D005314:Embryonic and Fetal Development; D005260:Female; D015723:Gene Library; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D013480:Superovulation
D017931:DNA Primers; D015342:DNA Probes; D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D012313:RNA