Efficient and fast targeted production of murine models based on ENU mutagenesis.

Mice with targeted genetic alterations are the most effective tools for deciphering organismal gene function. We generated an ENU-based parallel C3HeB/FeJ sperm and DNA archive characterized by a high probability to identify allelic variants of target genes as well as high efficiencies in allele retrieval and model revitalization. Our archive size of over 17,000 samples contains approximately 340,000 independent alleles (20 functional mutations per individual sample). Based on an estimated number of approximately 30,000 mouse genes, the parallel sperm/DNA archive should permit the identification and recovery of ten or more alleles per average target gene which translates to a calculated 99% success rate in the discovery of five allelic variants for any given average gene. The low rate of unrelated ENU-induced passenger mutations has no practical impact on the analysis of the allele-specific phenotype at the G3 generation because of dilution and free segregation of such unrelated passenger mutations. To date 39 mouse models representing 33 different genes have been recovered from our archive using in vitro fertilization techniques. The generation time for a murine model heterozygous for a mutation in a gene of interest is less than 2 months, i.e., three to four times faster compared with current embryonic stem-cell-based technologies. We conclude that ENU-based targeted mutagenesis is a powerful tool for the fast and high-throughput production of murine gene-specific models for biomedical research.

The dominant alopecia phenotypes Bareskin, Rex-denuded, and Reduced Coat 2 are caused by mutations in gasdermin 3.

Reduced Coat 2 (Rco2) is an ENU-induced mutation affecting hair follicle morphogenesis by an abnormal and protracted catagen. We describe chromosomal mapping and molecular identification of the autosomal dominant Rco2 mutation. The Rco2 critical region on mouse chromosome 11 encompasses the alopecia loci, Bareskin (Bsk), Rex-denuded (Re(den)), Recombination induced mutation 3 (Rim3), and Defolliculated (Dfl). Recently, the gasdermin (Gsdm) gene was described as predominantly expressed in skin and gastric tissues. We provide evidence for a murine-specific gene cluster consisting of Gsdm and two closely related genes which we designate as Gsdm2 and Gsdm3. We show that Gsdm3 reflects a mutation hotspot and that Gsdm3 mutations cause alopecia in Rco2, Re(den), and Bsk mice. We infer a role of Gsdm3 during the catagen to telogen transition at the end of hair follicle morphogenesis and the formation of hair follicle-associated sebaceous glands.

Synergistic interaction of two independent genetic loci causes extreme elevation of serum IgA in mice.

Understanding the molecular regulation of immunoglobulin A (IgA) expression is important as it plays an essential role in the first-line defence through mucosal secretions. Using inbred mouse strains, we identified two independent and dominant acting genetic loci that synergistically cause a 40-fold upregulation in serum IgA levels when introduced into the murine strain C57Bl/6J (B6). The first locus on chromosome 12 appears to be mainly responsible for the natural four-fold higher IgA levels in C3HeB/FeJ (C3H) compared to B6 mice. A second independent, chemically induced mutation on chromosome 5 caused a two-fold elevation when transferred from C3H into B6 mice. Both loci in concert effect a 40-fold elevation against the B6 genetic background. We determined the chromosomal localization of the two loci simultaneously by a one-step mapping process. The chemically induced mutation was identified within the immunoglobulin joining chain (IgJ) gene on chromosome 5. The major serum IgA modifier between the C3H and B6 was located on chromosome 12. This modifier region was mapped to a 350 kb region containing several immunoglobulin heavy-chain genes and the Ig alpha germline switch gene. We speculate that by interfering with both IgA expression and distribution, synergistic regulation of IgA is achieved.

Eomesodermin is required for mouse trophoblast development and mesoderm formation.

The earliest cell fate decision in the mammalian embryo separates the extra-embryonic trophoblast lineage, which forms the fetal portion of the placenta, from the embryonic cell lineages. The body plan of the embryo proper is established only later at gastrulation, when the pluripotent epiblast gives rise to the germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene Eomesodermin performs essential functions in both trophoblast development and gastrulation. Mouse embryos lacking Eomesodermin arrest at the blastocyst stage. Mutant trophoectoderm does not differentiate into trophoblast, indicating that Eomesodermin may be required for the development of trophoblast stem cells. In the embryo proper, Eomesodermin is essential for mesoderm formation. Although the specification of the anterior-posterior axis and the initial response to mesoderm-inducing signals is intact in mutant epiblasts, the prospective mesodermal cells are not recruited into the primitive streak. Our results indicate that Eomesodermin defines a conserved molecular pathway controlling the morphogenetic movements of germ layer formation and has acquired a new function in mammals in the differentiation of trophoblast.

Structure and regulation of the salivary gland secretion protein gene Sgs-1 of Drosophila melanogaster.

The Drosophila melanogaster gene Sgs-1 belongs to the secretion protein genes, which are coordinately expressed in salivary glands of third instar larvae. Earlier analysis had implied that Sgs-1 is located at the 25B2-3 puff. We cloned Sgs-1 from a YAC covering 25B2-3. Despite using a variety of vectors and Escherichia coli strains, subcloning from the YAC led to deletions within the Sgs-1 coding region. Analysis of clonable and unclonable sequences revealed that Sgs-1 mainly consists of 48-bp tandem repeats encoding a threonine-rich protein. The Sgs-1 inserts from single lambda clones are heterogeneous in length, indicating that repeats are eliminated. By analyzing the expression of Sgs-1/lacZ fusions in transgenic flies, cis-regulatory elements of Sgs-1 were mapped to lie within 1 kb upstream of the transcriptional start site. Band shift assays revealed binding sites for the transcription factor fork head (FKH) and the factor secretion enhancer binding protein 3 (SEBP3) at positions that are functionally relevant. FKH and SEBP3 have been shown previously to be involved in the regulation of Sgs-3 and Sgs-4. Comparison of the levels of steady state RNA and of the transcription rates for Sgs-1 and Sgs-1/lacZ reporter genes indicates that Sgs-1 RNA is 100-fold more stable than Sgs-1/lacZ RNA. This has implications for the model of how Sgs transcripts accumulate in late third instar larvae.

Cloning, chromosomal location, and expression analysis of murine Smarce1-related, a new member of the high-mobility 365 group gene family.

Proteins containing high-mobility group (HMG) domains are segregated into two major groups. Members of one group are identified by the presence of more than one HMG domain that binds to DNA without sequence specificity, and they are usually ubiquitously expressed. In contrast, members of the other group possess a single HMG domain with high affinity to specific DNA sequences. Generally, members of the second group resemble classic tissue-specific transcriptional regulators. In contrast, Smarce1/BAF-57 is a ubiquitously expressed, novel protein with a single HMG domain that displays nonspecific DNA-binding characteristics. Additionally, as a core subunit of the mammalian SWI/SNF-like transcriptional activator complex, Smarce1/BAF-57 is also the first member of the HMG protein family that was reported to contain a kinesin-like coiled-coil (KLCC) domain. Here we report the cloning, as well as the chromosomal and phylogenetic analysis, of a novel mammalian protein that is structurally related to Smarce1, termed Smarce1-related (Smarce1r). The unique arrangement of an HMG with a KLCC domain shared with Smarce1/BAF-57 suggests a similar, albeit still unknown, function in chromatin assembly as part of a mammalian SWI/SNF-like complex. The linkage of a single nonspecific DNA-binding HMG domain with a KLCC domain makes both proteins the founding members of a third group of HMG proteins.

Mouse beta-defensin 3 is an inducible antimicrobial peptide expressed in the epithelia of multiple organs.

One component of host defense at mucosal surfaces is epithelium-derived peptides with antimicrobial activity called defensins. We describe in this report the isolation and characterization of a murine homologue of human beta-defensin 2 (hBD-2) called mouse beta-defensin 3 (mBD-3). The predicted amino acid sequence shows the hallmark features of other known epithelial defensins, including the ordered array of six cysteine residues. Analysis of a genomic clone of mBD-3 revealed two exons separated by a 1.7-kb intron. The mBD-3 gene is localized at the proximal portion of chromosome 8, the site where genes for mouse alpha- and beta-defensins are found. Under basal condition, mBD-3 transcripts were detected at low levels in epithelial cells of surface organs, such as the intestine and lung. After instillation of Pseudomonas aeruginosa PAO1 into mouse airways, mBD-3-specific mRNA was upregulated significantly not only in large airways but also in the small bowel and liver. Recombinant mBD-3 peptide, produced from a baculovirus expression system, showed antimicrobial activity against P. aeruginosa PAO1 (MIC of 8 micrograms/ml) and Escherichia coli D31 (MIC of 16 micrograms/ml) in a salt-dependent manner. This study demonstrates that a murine homologue of hBD-2 is present in the respiratory system and other mucosal surfaces. These similarities between murine and human host defense apparatus provide further impetus to evaluate the mouse as a model for studying the human innate host defense system.

Construction of gene targeting vectors from lambda KOS genomic libraries.

We describe a highly redundant murine genomic library in a new lambda phage, lambda knockout shuttle (lambda KOS) that facilitates the very rapid construction of replacement-type gene targeting vectors. The library consists of 94 individually amplified subpools, each containing an average of 40,000 independent genomic clones. The subpools are arrayed into a 96-well format that allows a PCR-based efficient recovery of independent genomic clones. The lambda KOS vector backbone permits the CRE-mediated conversion into high-copy number pKOS plasmids, wherein the genomic inserts are automatically flanked by negative-selection cassettes. The lambda KOS vector system exploits the yeast homologous recombination machinery to simplify the construction of replacement-type gene targeting vectors independent of restriction sites within the genomic insert. We outline procedures that allow the generation of simple and more sophisticated conditional gene targeting vectors within 3-4 weeks, beginning with the screening of the lambda KOS genomic library.

Structural and phylogenetic characterization of human SLURP-1, the first secreted mammalian member of the Ly-6/uPAR protein superfamily.

Members of the Ly-6/uPAR protein family share one or several repeat units of the Ly-6/uPAR domain that is defined by a distinct disulfide bonding pattern between 8 or 10 cysteine residues. The Ly-6/uPAR protein family can be divided into two subfamilies. One comprises GPI-anchored glycoprotein receptors with 10 cysteine residues. The other subfamily includes the secreted single-domain snake and frog cytotoxins, and differs significantly in that its members generally possess only eight cysteines and no GPI-anchoring signal sequence. We report the purification and structural characterization of human SLURP-1 (secreted mammalian Ly-6/uPAR related protein 1) from blood and urine peptide libraries. SLURP-1 is encoded by the ARS (component B)-81/s locus, and appears to be the first mammalian member of the Ly-6/uPAR family lacking a GPI-anchoring signal sequence. A phylogenetic analysis based on the SLURP-1 primary protein structure revealed a closer relationship to the subfamily of cytotoxins. Since the SLURP-1 gene maps to the same chromosomal region as several members of the Ly-6/uPAR subfamily of glycoprotein receptors, it is suggested that both biologically distinct subfamilies might have co-evolved from local chromosomal duplication events.

Identification of a mammalian angiopoietin-related protein expressed specifically in liver.

Based on searches of EST databases for signal sequences and amphipathic helices, we have identified and cloned an angiopoietin-like gene, ANGPTL3. Multiple tissue Northern blots show that ANGPTL3 is expressed principally in the liver. ANGPTL3 is expressed early during liver development, and expression is maintained in adult liver. Human ANGPTL3 is a 460-amino-acid polypeptide with the characteristic structure of angiopoietins: a signal peptide, an extended helical domain predicted to form dimeric or trimeric coiled-coils, a short linker peptide, and a globular fibrinogen homology domain (FHD). Murine ANGPTL3 is a 455-acid polypeptide encoded by seven exons on mouse chromosome 4, spanning about 11 kb of DNA. ANGPTL3 contains the four conserved cysteines implicated in the intramolecular disulfide bonds within the FHD, but it does not contain two other cysteines that are found within the FHD of angiopoietins 1, 2, and 4. ANGPTL3 also does not contain the characteristic calcium binding motif found in the other angiopoietins. By radiation hybrid mapping and the use of surrounding genes, human ANGPTL3 maps to the 1p31 region.

A combined analysis of genomic and primary protein structure defines the phylogenetic relationship of new members if the T-box family.

T-box genes form an ancient family of putative transcriptional regulators characterized by a region of homology to the DNA-binding domain of the murine Brachyury (T) gene product. This T-box domain is conserved from Caenorhabditis elegans to human, and mutations in T-box genes have been associated with developmental defects in Drosophila, zebrafish, mice, and humans. Here we report the identification of three novel murine T-box genes and an investigation of their evolutionary relationship to previously known family members by studying the genomic structure of the T-box. All T-box genes from nematodes to humans possess a characteristic central intron that presumably was inherited from a common ancestral precursor. Two additional intron positions are also conserved with the exception of two nematode T-box genes. Subsequent intron insertions, potential deletions, and/or intron sliding formed a structural basis for the divergence into distinct subfamilies and a substrate for length variations of the T-box domain. In mice, the 11 T-box genes known to date can be grouped into seven subfamilies. Genes assigned to the same subfamily by genomic structure show related expression patterns. We propose a model for the phylogenetic relationships within the gene family that provides a rationale for classifying new T-box genes and facilitates interspecific comparisons.