Ernst-Martin Füchtbauer

Establishment of a gene-trap sequence tag library to generate mutant mice from embryonic stem cells

Establishment of a gene-trap sequence tag library to generate mutant mice from embryonic stem cells

A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome

A major challenge of the postgenomic era is the functional characterization of every single gene within the mammalian genome. In an effort to address this challenge, we assembled a collection of mutations in mouse embryonic stem (ES) cells, which is the largest publicly accessible collection of such mutations to date. Using four different gene-trap vectors, we generated 5,142 sequences adjacent to the gene-trap integration sites (gene-trap sequence tags; http://genetrap.de) from >11,000 ES cell clones. Although most of the gene-trap vector insertions occurred randomly throughout the genome, we found both vector-independent and vector-specific integration “hot spots.” Because >50% of the hot spots were vector-specific, we conclude that the most effective way to saturate the mouse genome with gene-trap insertions is by using a combination of gene-trap vectors. When a random sample of gene-trap integrations was passaged to the germ line, 59% (17 of 29) produced an observable phenotype in transgenic mice, a frequency similar to that achieved by conventional gene targeting. Thus, gene trapping allows a large-scale and cost-effective production of ES cell clones with mutations distributed throughout the genome, a resource likely to accelerate genome annotation and the in vivo modeling of human disease.

cDermo-1 expression indicates a role in avian skin development.

Basic helix-loop-helix (bHLH) transcription factors have been shown to be important regulatory proteins for tissue determination and differentiation. We cloned the chicken homologue of the gene of the murine Twist-related bHLH protein Dermo-1, which we named cDermo-1, and analyzed its sequence and embryonic expression. Our sequence data suggest a decisive role of Dermo-1 proteins in the evolution of amniote skin. We present a detailed analysis of cDermo-1 expression during avian embryonic development. cDermo-1 is first expressed in a variety of mesodermal tissues of the chick embryo including the limb buds, but later becomes restricted to the subectodermal mesenchyme of the integument and the developing feather buds, indicating a role of cDermo-1 during avian skin and feather development.

Dmdmdx-βgeo: A new allele for the mouse dystrophin gene

During a gene trap screen, an insertion of the gene trap vector into the dystrophin gene, creating a new allele for the Dmd gene, has been discovered. Because the ROSAβgeo vector was used, the new allele is called Dmdmdx‐βgeo. The insertion occurred 3′ of exon 63 of the dystrophin gene, resulting in a mutation that affects all presently known dystrophin isoforms. In contrast to spontaneous or ENU‐induced alleles, Dmdmdx‐βgeo can be used to follow dystrophin expression by staining for β‐galactosidase activity. The high sensitivity of this method revealed additional and earlier expression of dystrophin during embryogenesis than that seen previously with other methods. Dystrophin promoters are active predominantly in the dermamyotome, limb buds, telencephalon, floor plate, eye, liver, pancreas anlagen, and cardiovascular system. Adult Dmdmdx‐βgeo mice show reporter gene expression in brain, eye, liver, pancreas, and lung. In skeletal and heart muscle, β‐galactosidase activity is not detectable, confirming Western blot data that indicate the absence of the mutant full‐length protein in these tissues. Hemizygous Dmdmdx‐βgeo mice show muscular dystrophy with degenerating muscle fibers, cellular infiltration, and regenerated muscle fibers that have centrally located nuclei. Some mutant animals develop a dilated esophagus, probably due to constriction by the hypertrophic crura of the diaphragm. Dev. Dyn. 1998;212:229–241.

MAID : A MATERNALLY TRANSCRIBED NOVEL GENE ENCODING A POTENTIAL NEGATIVE REGULATOR OF BHLH PROTEINS IN THE MOUSE EGG AND ZYGOTE

We isolated an abundant novel cDNA SSEC‐8 from a subtraction cDNA library enriched for maternal transcripts that are still present in the mouse 2 cell stage embryo. This gene is evolutionarily conserved and maps to the distal region of mouse chromosome 2. The deduced polypeptide sequence of the encoded protein contains a conserved helix‐loop‐helix (HLH) motif without a basic DNA binding domain, suggesting that it functions as a negative regulator of basic (b) HLH transcription factors. Gel mobility shift assays show that in vitro translated protein prevents the E12/MyoD bHLH dimer from binding to DNA. Also, transient overexpression of this protein in C2C12 cells reduced the transcription of a CAT‐reporter regulated by an E12/MyoD driven enhancer. The 3′‐UTR contains consensus sequences of cytoplasmic polyadenylation elements (CPEs), and the length of its poly (A) tail changes during oocyte maturation, indicating that its expression is controlled by timely activation of translation. This new gene, Maid, models the translational and transcriptional regulation of gene expression during the transition from gamete to embryo. Dev. Dyn. 209:217–226, 1997.

ANGIOTENSIN II STIMULATES CONTRACTILITY AND C-FOS GENE EXPRESSION IN ISOLATED ATRIAL HUMAN MYOCARDIUM

The renin-angiotensin system plays an important role in the pathogenesis of many cardiovascular disorders. Althougth the acute effect of angiotensin II on myocardial contractility, as well as the chronic effect of angiotensin II on cardiac growth, has been studied in several species, little is known about these effects in human myocardium.