High-throughput mouse knockouts provide a functional analysis of the genome.
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Animals↗
Biomedical subjects
R Ramirez-Solis
Publications and source records attributed to R Ramirez-Solis.
Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN.
A high-throughput, retrovirus-mediated mutagenesis method based on gene trapping in embryonic stem cells was used to identify a novel mouse gene. The human ortholog encodes a transmembrane protein containing five extracellular immunoglobulin-like domains that is structurally related to human NEPHRIN, a protein associated with congenital nephrotic syndrome. Northern analysis revealed wide expression in humans and mice, with highest expression in kidney. Based on similarity to NEPHRIN and abundant expression in kidney, this protein was designated NEPH1 and embryonic stem cells containing the retroviral insertion in the Neph1 locus were used to generate mutant mice. Analysis of kidney RNA from Neph1(-/-) mice showed that the retroviral insertion disrupted expression of Neph1 transcripts. Neph1(-/-) pups were represented at the expected normal Mendelian ratios at 1 to 3 days of age but at only 10% of the expected frequency at 10 to 12 days after birth, suggesting an early postnatal lethality. The Neph1(-/-) animals that survived beyond the first week of life were sickly and small but without edema, and all died between 3 and 8 weeks of age. Proteinuria ranging from 300 to 2,000 mg/dl was present in all Neph1(-/-) mice. Electron microscopy demonstrated NEPH1 expression in glomerular podocytes and revealed effacement of podocyte foot processes in Neph1(-/-) mice. These findings suggest that NEPH1, like NEPHRIN, may play an important role in maintaining the structure of the filtration barrier that prevents proteins from freely entering the glomerular urinary space.
Amino Acid Sequence↗
Modifying the mouse: design and desire.
Genetic modification of endogenous genes in mice has become possible by applying gene targeting techniques to embryonic stem (ES) cells and using specific clones of cells to generate mice. Despite the experimental opportunities offered by the creation of organisms with specific genetic changes, there are considerable technical obstacles which can confound the routine implementation of this technology. This review addresses some recent advances in the ability to construct mice with a variety of genetic modifications. These include an increased understanding of the basic cell biology and in vitro growth characteristics of ES cells, which has facilitated germ line transmission of manipulated clones on a routine basis. The techniques that are used to isolate "targeted" clones of ES cells have been summarized, and the current status of strategies which have been successfully used to make very specific modifications of the genome are discussed.
Alleles↗