ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 223, No. 1, May, pp. 140-148, 1983 Cloning of Total mRNA Populations from Adult and Embryonic Mice MICHAEL J. TOCCI,’ KENNETH A. FLEMING,2 AND JOHN J. MONAHAN Roche In-stitute of Molecular Biology, Department of Cell Biology, Nutley, New Jersey 07110 Received November 18, 1982 Total clone banks of cDNAs synthesized from poly(A)-RNA obtained from three stages of the developing mouse were constructed. The stages chosen were 13-day-old embryo, neonatal, and fully grown adult. To have as complete a bank as possible, large numbers of individual clones were generated -400,000 for the 13th day embryo and neonatal mouse and -610,000 for the adult bank. In each case the clone bank was constructed by inserting double stranded cDNA into the PstI site of pBR322 by the “G-C tailing” method. Sequences cloned in this way could be separated from the plasmid host DNA by treatment of the resultant total chimeric plasmid population with PstI. Aliquots of the cloned cDNA material were labeled with 32P by “nick translation” using Escherichia coli DNA polymerase I for the preparation of hybridization probes. Back- hybridization of these probes to the total clone banks allowed the determination of the sequence diversity among the above three very different developmental stages. The use of such clone banks should allow the identification of developmental stage specific mRNAs. Very little is known about how growth and development is controlled at the mo- lecular level in developing animals. The continuously changing nature (as the em- bryo develops) of the source material often has made even superficial molecular anal- ysis difficult. Studies in the past were often confined to the measurements of rates of RNA synthesis during embryogenesis, the content of RNA/cell, or the effects of RNA inhibitors on the process of embryogene- sis. Recently, some progress has been made in the isolation and analysis of poly(A)- RNA from early mouse embryos using classical techniques (l-5). Quantitative as well as qualitative differences were seen as the embryo developed. However, fur- ’ Present address: Department of Biochemical Ge- netics, P.O. Box 2000, Merck Sharpe & Dohme Re- search Laboratories, Rahway, New Jersey 07065. ’ Present address: University of Oxford, Depart- ment of Pathology, John Radcliffe Hospital, Head- ington, Oxford, 0X3, 9DU, England. 3 To whom all correspondence should be addressed. ther progress along these lines will prob- ably be slow because of the limited supply of starting material and the monumental amount of work involved in carrying out such studies. The application of recombinant DNA techniques has brought new hope to the possibility of unraveling the complex pro- cess of multigene expression during em- bryogenesis. With this technology specific RNA species can be obtained and at least some attempt can be directed toward searching for nearby regulatory DNA se- quences. Perhaps equally significant, clon- ing methodology provides an essentially unlimited supply of cloned DNA from a small supply of initial starting material. For a study of mammalian embryogenesis and development often only minute quan- tities of material can be isolated practi- cally. We have chosen the mouse embryo as a model system for studying the mo- lecular biology of embryogenesis. It is well characterized in terms of the systems morphological development, embryogene- 0003-9861/83 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved 140