Gail R. Martin

This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures, derived from isolated single cells, can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells, or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo, including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development.

We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of low-abundance mRNAs. In essence, cDNAs are generated by using the DNA polymerase chain reaction technique to amplify copies of the region between a single point in the transcript and the 3' or 5' end. The minimum information required for this amplification is a single short stretch of sequence within the mRNA to be cloned. Since the cDNAs can be produced in one day, examined by Southern blotting the next, and readily cloned, large numbers of full-length cDNA clones of rare transcripts can be rapidly produced. Moreover, separation of amplified cDNAs by gel electrophoresis allows precise selection by size prior to cloning and thus facilitates the isolation of cDNAs representing variant mRNAs, such as those produced by alternative splicing or by the use of alternative promoters. The efficacy of this method was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is approximately 2.9 kilobases. After less than 0.05% of the cDNAs produced had been screened, 29 independent int-2 clones were isolated. Sequence analysis demonstrated that the 3' and 5' ends of all four int-2 mRNAs were accurately represented by these clones.

Evidence is accumulating that members of the FGF gene family provide signals that act locally to regulate growth and patterning in vertebrate embryos. In this report, we provide a detailed analysis of the mouse Fgf8 gene. We have mapped the Fgf8 locus to the distal region of mouse chromosome 19, and sequenced the 5' coding region of the gene. Our data identify a new coding exon, and locate multiple splice donor and splice acceptor sites that can be used to produce at least seven transcripts encoding a family of secreted FGF8 proteins with different N termini. From these results, it appears that Fgf8 is structurally the most complex member of the FGF family described to date. In the embryo, many of the regions in which Fgf8 RNA is localized are known to direct outgrowth and patterning, including the apical ectodermal ridge of the limb bud, the primitive streak and tail bud, the surface ectoderm overlying the facial primorida and the midbrain-hindbrain junction, suggesting that FGF8 may be a component of the regulatory signals that emanate from these regions.