This cross resulted in normal litter sizes and Mendelian distributions of genotypes. (N-myc downstream-regulated gene), which is highly expressed in brain and heart. Previous studies showed that mutant alleles that exhibit several development defects different from those previously reported. Our homozygous mice showed growth Dantrolene sodium Hemiheptahydrate retardation and postnatal lethality. Spleen and thymuses of mice are considerably reduced in size from 3 weeks of age. Histological analysis revealed abnormal hyperkeratosis in the squamous foregut and abnormal loss of erythrocytes in the spleen of mice. In addition, we observed an abnormal hind limb clasping phenotype upon tail suspension suggesting neurological abnormalities. Consistent to these abnormalities, Ndrg4 is expressed in smooth muscle cells of the stomach, macrophages of the spleen and neurons. Availability of the conditional allele for should facilitate further detailed analyses of the potential roles of Ndrg4 in gut development, nervous system and immune system. . in mice resulted in vertebral homeotic transformations in thoracic/lumbar and lumbar/sacral transitional regions in a dose-dependent manner . An gene. When a global null mutation for the gene was generated from the floxed mice, to our surprise, we observed several more severe phenotypes different from those previously reported: an abnormal hind limb clasping phenotype and growth retardation with disproportional small spleens and thymuses from the age of about 3 weeks. In addition, our homozygous mutant mice also exhibited partial postnatal lethality. In agreement with reduced size in the forestomach and the spleen of mutant mice, we observed abnormal hyperkeratosis in the squamous foregut and abnormal loss of erythrocytes in the spleen of mice. We also detected a considerable Ndrg4 expression in stomach, spleen and neurons of wt mice. 2. Methods 2.1. Mouse Breeding and Generation of Mutant Alleles All strains were maintained on a mixed 129 and C57/BL6 background. The animals were handled in accordance with institutional guidelines with the approval of the Institutional Animal Care and Use Committee of Vanderbilt University School of Medicine. The authors are currently exploring possibilities to deposit the newly developed mice to public repositories and are happy to share them upon request. To identify tissue-specific functions of Ndrg4, we have created a conditional allele of the mouse gene by introducing Cre recombinase recognition sites (locus. This strategy involved introducing a site and a neomycin resistance cassette within the intron 5, and another site into the intron 7 (Fig. Dantrolene sodium Hemiheptahydrate 1a), such that Cre recombination excises the exon 5-7 Rabbit Polyclonal to SLC39A1 leading reading frame shift. A floxed targeting vector was constructed based Dantrolene sodium Hemiheptahydrate Dantrolene sodium Hemiheptahydrate on the 129-Sv mouse genomic fragment. A 744bp exons 6-8 was inserted into the Floxed sites and a cassette containing the herpes simplex virus thymidine kinase gene expressed from the phosphoglycerate kinase promoter (transgenic mice to generate heterozygous mice for the floxed allele without neo (mutant mice (knockout mice, the mice were crossed with transgenic mice, which express the Cre recombinase in germ cells . Cre-dependent deletion was detected with primers P1, P2 and P3 (5-GCTCCCACTCCAATGCCAATC-3) (Fig. 1a, c). The resulting mutant mice. Open in a separate window Fig. 1 Generation of mutant lines. (a) Schematic representation of the targeting strategy and deleting exons 5-7 in mice. Top: genomic structure. The targeting vector was designed so that Dantrolene sodium Hemiheptahydrate sites would flank the 744bp exons 5-7. Homologous recombination with the targeting vector generated the allele; Flp deletion created the floxed allele; subsequent Cre deletion led to the deletion allele. Blue boxes, exons; red boxes, external probes for Southern blot analysis (diagnostic band lengths pre/post-recombination indicated); neo, Neomycin resistance cassette; TK, thymidine kinase cassette. Red and green arrowheads indicate and sites, respectively. A, is abundantly expressed in the murine brain and heart [1,3,18] and that is essential for normal cardiac development in zebra fish . To elucidate the effects of deficiency and allow temporal and tissue-specific gene inactivation, we have.