CONSTRUCTION OF A MOUSE BLASTOCYST CDNA LIBRARY BY PCR AMPLIFICATION FROM TOTAL RNA

Studies of the development and differentiation of early mammalian embr yos have been severely limited by the paucity of material. Such studie s have been largely restricted to the examination of abundant genes/pr oteins or to developmental expression studies of known genes for which DNA sequence data are available, allowing the use of reverse transcri ption and polymerase chain reaction amplification (RT-PCR). To elimina te the need for hundreds or thousands of oocytes or embryos in the con struction of representative cDNA libraries, we describe a technique fo r generating and cloning cDNA using small caesium chloride gradient ce ntrifugation to isolate total RNA from oocytes or embryos, followed by RT-PCR of mRNA from this total RNA. Total RNA was isolated from 70 mo use 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 plasmino gen activator, and B1 and B2 repetitive elements in frequencies compar able 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 sequen ce 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. (C) 1996 Wiley-Liss, Inc.