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 lambda gt11. 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 X 10(6) 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 beta-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 (less than 1% of mRNA) RNAs.
Weng
D E
DE
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Morgan
R A
RA
Gearhart
J D
JD
D000199:Actins / Q000235:genetics; D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA; D015870:Gene Expression; D007426:Genes, Intracisternal A-Particle; D015698:Genomic Library; D006657:Histones / Q000235:genetics; D007633:Keratins / Q000235:genetics; D051379:Mice; D008813:Mice, Inbred ICR; D012333:RNA, Messenger / Q000378:metabolism*
D000199:Actins; D006657:Histones; D012333:RNA, Messenger; D007633:Keratins; D004247:DNA