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Component 7 of the BayGenomics project is led by Dr. Pao-Tien Chuang, Assistant Professor of Biochemistry and Biophysics at UCSF. Component 7 is producing several knockout mice each year in order to identify new genes that have important roles in the embryonic development of the heart and lung. We are focusing on novel genes with informative sequences and/or genes with restricted spatial or temporal expression patterns in the cardiopulmonary system. If the knockout mice manifest embryonic lethality, homozygous embryos are collected and analyzed at different stages of embryonic development. We are performing extensive histologic studies to assess developmental abnormalities, in situ hybridization and antibody staining to understand the molecular defects, and microarray studies to uncover signaling networks that may be perturbed in the mutant mice. For homozygous animals that do not exhibit any gross developmental phenotypes, adult mice are subjected to physiological studies in order to determine if cardiovascular or pulmonary function has been perturbed. A complete characterization of these knockout mice may require more extensive genetic manipulations (e.g., production of conditional knockout mice or transgenic mice for overexpression studies).
Recent progess includes the purchase and installation of equipment necessary to fulfill the aims outlined above. The major equipment purchased is a Nikon SMZ 1500 steroscope/DXM1200 digital camera for dissecting mouse embryos and capturing images.
Together with Component 6, we have selected seven gene-trapped lines (see table below) based on their in-situ hybridization data generated in Component 3, as well as sequence analysis generated in Component 1 and 2, for blastocyst injection (by Component 1) in order to generate knockout mice. It is well established that in most cases tissue-specific expression of genes implicates that these genes play an essential role in the development and/or function of these tissues. In addition, cell-cell interaction plays a key role in the development of various tissue/organs. For instance, it is well known that in the developing lung, interactions between the epithelium and the surrounding mesenchyme play a key role in inducing lung branching in a temporal- and spatial-specific manner. Therefore, genes expressed in either the lung epithelium or mesenchyme will be good candidates for mediating the cross-talk between the epithelium and the mesenchyme. NST035 is primarily expressed in the epithelium of the lung. XST087 is ubiquitously expressed throughout the lung and faintly in a few other tissues as well. XB677 is strongly expressed in the epithelium of the lung with faint staining throughout the embryo. KST288/KST273 (two trapped lines of the same gene) is expressed in the neural tube, the gut endoderm, and the epithelium of the lung. EX177 is expressed only in the bone of the developing embryo where it is faintly expressed in the vertebral column but strongly expressed in the limbs.
All seven injected gene-trapped lines gave rise to multiple high-percentage male chimeras. These male chimeras were mated to Black SW females to test coat color and germline transmission of the gene-trapped allele. As summarized in the table below, we have obtained germline transmission from two gene-trapped lines and are in the process of testing the other five. These mice will help elucidate the role that these genes may play in cardiopulmonary development and diseases.
Gene
Gene-Trapped Lines injected
High percentage male chimeras
Transmission of Coat color through male chimeras
Germ Line Transmission of gene-trapped allele
Similar to Rat amino acid A transporter mRNA
NST035
Yes
Yes
Yes
Novel- similar to human GOLPH2
KST288/KST273
Yes
Yes
Yes
Novel-similar to human Protein Tyrosine Kinase 7
XST087
Yes
Being tested
Novel-actin dependent regulator of chromatin, matrix associated
XB677
Yes
Being tested
Novel-cell adhesion molecule related
EX177
Yes
Being tested
Novel-similar to membrane glycoprotein
GST042
Yes
Being tested