Non-redundant functions of the mouse Ssrp1 and Pfdn1 genes revealed by gene targeting and gene entrapment

Abstract

NON-REDUNDANT FUNCTIONS OF THE MOUSE SSRP1 AND PFDN1 GENES REVEALED BY GENE TARGETING AND GENE ENTRAPMENT

SHANG CAO

Dissertation under the direction of Professor Henry Earl Ruley In the post-genome era, gene targeting and gene entrapment provide efficient strategies to analyze gene functions in mice. Conventional gene targeting ablates individual genes by homologous recombination while gene entrapment generates large numbers of disrupted genes from which specific mutations are selected for further studies. Mouse Ssrp1 and Pfdn1 gene were disrupted in murine embryonic stem cells, by conventional gene targeting and gene entrapment, respectively, and were subsequently introduced into the germ line. The Ssrp1 gene encodes a high-mobility-group (HMG) protein SSRP1, a member of a conserved chromatin-remodeling complex (FACT/DUF/CP) implicated in DNA replication, transcription and repair. Ssrp1 homozygous mutant embryos die soon after implantation, and pre-implantation blastocysts are defective for cell outgrowth and/or survival in vitro. Further loss of p53 does not influence growth and/or survival defects caused by loss of Ssrp1 function. Thus, Ssrp1 appears to encode non-redundant and p53-independent functions that are essential for cell viability. The Pfdn1 gene, coding for the first subunit of molecular chaperone prefoldin, was ablated by insertion of a gene trap retrovirus (GTR1.3). Null mutant mice are born at nearly Mendelian ratios, but most die before 5 weeks of age, after a period of stunted growth, physical wasting and neuromuscular abnormalities. Postnatal phenotypes of homozygous mutant mice include: (1) defective commissures in the central nervous system (CNS);(2) hematopoietic defects consisting of micronucleated erythrocytes and neutropils with abnormal nuclear segmentation; and (3) lymphopoietic defects include loss of B cell precursors and altered ratios of T cell subpopulations. Fibroblasts from mutant embryos (MEFs) are viable but display abnormal cytoskeletal organization, suggesting that Pfdn1 encodes functions required for cytoskeletal assembly. Thus, Pfdn1appears to encode non-redundant functions required for mouse development and survival, presumably by affecting cytoskeleton formation.