RBBP7

Abstract: The technique of gene targeting allows for the introduction of engineered genetic mutations into a mouse at a determined genomic locus. The process of generating mouse models with targeted mutations was developed through both the discovery of homologous recombination and the isolation of murine embryonic stem cells (ES cells). Homologous recombination is a DNA repair mechanism that is employed in gene targeting to insert a designed mutation into the homologous genetic locus. Targeted homologous recombination can be performed in murine ES cells through electroporation of a targeting construct. These ES cells are totipotent and, when injected into a mouse blastocyst, they can differentiate into all cell types of a chimeric mouse. A chimeric mouse harboring cells derived from the targeted ES cell clone can then generate a whole mouse containing the desired targeted mutation. The initial step for the generation of a mouse with a targeted mutation is the construction of an efficient targeting vector that will be introduced into the ES cells.

Abstract: After the successful completion of the human genome project (HGP), biological research in the postgenome era urgently needs an efficient approach for functional analysis of genes. Utilization of knockout mouse models has been powerful for elucidating the function of genes as well as finding new therapeutic interventions for human diseases. Gene trapping and gene targeting are two independent techniques for making knockout mice from embryonic stem (ES) cells. Gene trapping is high-throughput, random, and sequence-tagged while gene targeting enables the knockout of specific genes. It has been about 20 years since the first gene targeting and gene trapping mice were generated. In recent years, new tools have emerged for both gene targeting and gene trapping, and organizations have been formed to knock out genes in the mouse genome using either of the two methods. The knockout mouse project (KOMP) and the international gene trap consortium (IGTC) were initiated to create convenient resources for scientific research worldwide and knock out all the mouse genes. Organizers of KOMP regard it as important as the HGP. Gene targeting methods have changed from conventional gene targeting to high-throughput conditional gene targeting. The combined advantages of trapping and targeting elements are improving the gene trapping spectrum and gene targeting efficiency. As a newly-developed insertional mutation system, transposons have some advantages over retrovirus in trapping genes. Emergence of the international knockout mouse consortium (IKMP) is the beginning of a global collaboration to systematically knock out all the genes in the mouse genome for functional genomic research.

Abstract: The YPT1 gene of the yeast Saccharomyces cerevisiae codes for a guanine nucleotide-binding protein which is essential for cell viability. Using as hybridization probe cloned yeast YPT1 gene sequences, we have isolated from cDNA libraries prepared from RNA of mouse F9 and C3H10T1/2 cells several overlapping cDNA clones with identical sequence in the regions of overlap. The cDNAs were derived from a gene, designated ypt1, which codes for a protein of 205 amino acids with 71% homology to the yeast YPT1 gene product. Amino acid sequences typical for guanine nucleotide-binding proteins and characteristic for ypt proteins are perfectly conserved in the mouse ypt1 protein. Two mRNAs of 1600 and 3200 nucleotides, originating from the mouse ypt1 gene and differing in the length of their 3'-non-translated region, were identified in mouse F9 cells and in all mouse tissues examined. A monoclonal antibody specifically recognizing the 23.5-kd yeast YPT1 protein cross-reacted with a protein of identical size on protein blots of mouse, rat, pig, bovine and human cell lines.

Abstract: During a search for obesity candidate genes in a small region of the mouse genome, we noticed that many genes when knocked out influence body weight. To determine whether this was a general feature of gene knockout or a chance occurrence, we surveyed the Jackson Laboratory Mouse Genome Database for knockout mouse strains and their phenotypes. Body weights were not available for all strains so we also obtained body weight information by contacting a random sample of investigators responsible for a knockout strain. We classified each knockout mouse strain as (1) lighter and smaller, (2) larger and heavier, or (3) the same weight, relative to control mice. We excluded knockout strains that died early in life, even though this type of lethality is often associated with a small embryo or reduced body size. Based on a dataset of 1,977 knockout strains, we found that that 31% of viable knockout mouse strains weighed less and an additional 3% weighed more than did controls. Body weight is potentially a latent variable in about a third of experiments that use knockout mice and should be considered in interpreting experimental outcomes, e.g., in studies of hypertension, drug and hormone metabolism, organ development, cell proliferation and apoptosis, digestion, heart rate, or atherosclerosis. If we assume that the knockout genes we surveyed are representative then upward of 6,000 genes are predicted to influence the size of a mouse. Body weight is highly heritable, and numerous quantitative trait loci have been mapped in mice, but "multigenic" is an insufficient term for the thousands of loci that could contribute to this complex trait.