Biomedical subjects

W C Skarnes

Publications and source records attributed to W C Skarnes.

26 records · Page 2Linked to original sources

PubMed1992

The gene trap approach in embryonic stem cells: the potential for genetic screens in mice.

The gene trap approach in embryonic stem cells was developed as a means to screen for genes expressed during early postimplantation development in the mouse. We have validated the approach by showing that lacZ from the integrated vector is activated by splicing to endogenous exons and expressed in embryos in patterns that mimic those of the endogenous genes. These insertions can produce developmental defects in homozygous mice. The results indicate that a large screen of gene trap cell lines on the basis of embryonic lacZ expression is feasible and should provide a new source of genes, mouse mutants and mouse strains that express lacZ in particular domains and lineages. The gene trap approach could be extended to a smaller screen for genes based on mutant phenotypes.

Amino Acid Sequence↗

PubMed1992

A gene trap approach in mouse embryonic stem cells: the lacZ reported is activated by splicing, reflects endogenous gene expression, and is mutagenic in mice.

We have confirmed that the gene trap vector pGT4.5 creates spliced fusion transcripts with endogenous genes and prevents the synthesis of normal transcripts at the site of integration. cDNA was prepared to the lacZ fusion transcript in three ES cell lines to recover endogenous exon sequences upstream of lacZ. Each of the clones detected a unique-sized endogenous transcript, as well as the fusion transcript in the ES cell line from which the clone was derived. Sequence analysis of these clones and larger clones isolated from a random-primed cDNA library showed that the splice acceptor was used properly. For two insertions, the expression patterns of the lacZ reporter and the associated endogenous gene were compared in situ at three embryonic stages and were found to be similar. Three gene trap insertions were transmitted into the germ line, and abnormalities were observed with two of the three insertions in the homozygous state. RNA obtained from mice homozygous for the two mutant gene trap insertions was analyzed for normal endogenous transcripts and negligible amounts were detected, indicating that little splicing around the gene trap insertion occurred. This work demonstrates the capacity of the gene trap vector to generate lacZ fusion transcripts, to accurately report endogenous gene expression, and to mutate the endogenous gene at the site of integration.

Amino Acid Sequence↗

PubMed1991

RNA polymerase II pauses in vitro, but does not terminate, at discrete sites in promoter-proximal regions on polyomavirus transcription complexes.

Many RNA polymerases stall and/or prematurely terminate transcription nearby the early and late promoters of polyomavirus in vivo during the late phase of productive infection. In this paper we analyzed the RNAs made when these promoter-proximal RNA polymerases were allowed to elongate their nascent chains in vitro on viral transcription complexes isolated from infected cells. RNA was labeled in the presence of a high specific activity of one [alpha-32P]-ribonucleoside triphosphate (rNTP) (less than 1 microM final concentration) and saturating concentrations of the other three rNTPs. Under these conditions, promoter-proximal RNAs of discrete sizes were produced. We show that these discrete RNA species are produced by pausing of RNA polymerase II due to limiting concentrations of one of the rNTPs, and that the positions of the pause sites depend on which rNTP is limiting. This pausing does not result in release of the RNA polymerase or the nascent RNA chain from the transcription complex, as these chains can be further extended when high concentrations of all four rNTPs are supplied. Our results conflict with the interpretations of other investigators who suggest that formation of discrete RNA products, under similar in vitro conditions, reflects authentic termination by RNA polymerase II.

DNA, Viral↗

PubMed1990

Entrapment vectors: a new tool for mammalian genetics.

The use of mouse embryonic stem (ES) cells has facilitated the creation of mouse strains carrying desired genetic alterations. Recently, "entrapment" vectors have been developed to identify and mutate genes active during embryogenesis. These vectors in combination with ES cells may provide an efficient screening strategy to find genes that control mouse development.

Animals↗

PubMed1989

Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes.

A strategy was devised for identifying regions of the mouse genome that are transcriptionally active in a temporally and spatially restricted manner during development. The approach is based on the introduction into embryonic stem cells of two types of lacZ reporter constructs that can be activated by flanking mouse genomic sequences. Embryonic stem cells containing the lacZ constructs were used to produce chimaeric mice. Developmental regulation of lacZ expression occurred at a high frequency. Molecular cloning of the flanking endogenous genes and introduction of these potential insertional mutations into the mouse germ line should provide an efficient means of identifying and mutating novel genes important for the control of mammalian development.

Animals↗

PubMed1989

Production of a mutation in mouse En-2 gene by homologous recombination in embryonic stem cells.

A full understanding of the function of genes that control developmental events can be obtained only by a combination of molecular and mutational analysis. One putative developmental gene is the mouse engrailed-like gene En-2, which was isolated by virtue of its extensive homology to Drosophila engrailed, which contributes to the control of segmentation in the developing insect. Our hybridization analysis in situ has revealed that expression of En-2 is restricted to a specific domain of the developing central nervous system from 8 days of development on, indicating a role for the gene in establishing spatial domains in the brain. Unfortunately no En-2 mutations are available to elucidate further its function in development. To this end, we report here the isolation of three pluripotent embryonic stem cell lines in which one copy of the homoeobox-containing gene, En-2, has been altered by homologous recombination.

Animals↗

PubMed1989

Nerve growth factor regulates gene expression by several distinct mechanisms.

To help elucidate the mechanisms by which nerve growth factor (NGF) regulates gene expression, we have identified and studied four genes (a-2, d-2, d-4, and d-5) that are positively regulated by NGF in PC12 cells, including one (d-2) which has previously been identified as a putative transcription factor (NGF I-A). Three of these genes, including d-2, were induced very rapidly at the transcriptional level, but the relative time courses of transcription and mRNA accumulation of each of these three genes were distinct. The fourth gene (d-4) displayed no apparent increase in transcription that corresponded to the increase in its mRNA, suggesting that NGF may regulate its expression at a posttranscriptional level. Thus, NGF positively regulates gene expression by more than one mechanism. These genes could also be distinguished on the basis of their response to cyclic AMP. The expression of d-2 and a-2 was increased by cholera toxin and further augmented by NGF; however, cholera toxin not only failed to increase the levels of d-5 and d-4 mRNA but also actually inhibited the NGF-dependent increase. The expression of each of these genes, including d-2 (NGF I-A), was also increased by fibroblast growth factor, epidermal growth factor (EGF), phorbol myristate acetate, and in some cases insulin, showing that the regulation of these genes is not unique to NGF. Because each of these genes was expressed in response to phorbol myristate acetate and EGF, their expression may be necessary but is certainly not sufficient for neurite formation. The protein kinase inhibitor K-252a prevented the NGF-associated, but not the acidic FGF-associated, induction of d-2 and d-5 gene expression, suggesting that these two growth factors may regulate gene expression via different cellular pathways. The study of the regulation of the expression of these and other NGF-inducible genes should valuable new information concerning how NGF and other growth factors cause neural differentiation.

Animals↗

PubMed1988

RNA polymerases stall and/or prematurely terminate nearby both early and late promoters on polyomavirus DNA.

Levels of transcription within the E and L strands of the five major PstI fragments of polyomavirus (strain AT3) were measured by pulse-labeling RNA both in infected cells and in isolated nuclei or viral transcription complexes during the late phase of infection. Quantification was assured by hybridization to single-stranded DNAs in solution followed by collection of hybrids on nitrocellulose filters and ribonuclease treatment. The level of in vivo transcription in the region of the early (E strand) promoter was two- to threefold higher than that in all other E-strand regions, suggesting that most RNA polymerases prematurely terminate transcription shortly downstream from this promoter during the late phase. In vitro transcription levels in this region were five- to tenfold higher than in the remainder of the E strand, suggesting that many RNA polymerases 'stall' shortly after initiation in vivo but can be reactivated and continue transcription in vitro upon exposure to detergents and high salt solution. Some premature termination nearby the late (L strand) promoter was also detected by the same method. Strikingly, many RNA polymerases also stalled on the L strand in the region of the early promoter, some 5 x 10(3) bases downstream from the late promoter. Treatment of cells with dichlororibofuranosylbenzimidazole did not affect polymerases that stalled or terminated prematurely, but strongly reduced the presence of polymerases that normally transcribed throughout the entire E or L strand. Examination of the size of RNA chains produced during in vitro incubations showed that many polymerases stalled in vivo within 50 to 100 nucleotides downstream from the initiation sites on both DNA strands. The number of polymerases active in vitro at the E strand promoter was similar to the number of polymerases at the L strand promoter. However, in contrast to L-strand transcription, most of the polymerases that initiated at the E-strand promoter were incapable of extended transcription in vivo. These results suggest that large T antigen-mediated repression of E-strand transcription is not simply due to the exclusion of RNA polymerases from the early promoter. Stalling and/or premature termination by RNA polymerases shortly downstream from the early promoter appears to be a mechanism by which temporal regulation of polyomavirus gene expression can be effected.

Base Sequence↗