PARENT SESSION
1:30 PM to 3:30 PM
Wednesday, April 25, 2001
Poster Session 24 DNA Damage and Repair II
Room: Exhibition Center

(P24-302) Mrad9 Knockout ES cells and Mice.

Hopkins, Kevin¹, Auerbach, Wojtek², Zheng, Wei¹, Hang, Haiying¹, Joyner, Alexandra², Lieberman, Howard¹, ^{1 2}

ABSTRACT-
We have cloned mouse genomic and cDNA orthologues of the fission yeast S. pombe cell cycle checkpoint control gene rad9. The rad9 gene of S. pombe is involved in the repair of damaged DNA caused by exposure to ionizing and ultraviolet light. S. pombe cells containing rad9::ura4⁺ are highly sensitive to ionizing radiation and ultraviolet light, and are unable to delay cycling in late G2 after exposure to radiation. Therefore, these cells lack G2/M checkpoint control. To determine the role of a mammalian version of this gene in radioresistance and cell cycle checkpoint function, we used the cloned Mrad9 gene to generate heterozygous Mrad9 deleted mouse embryonic stem (ES) cells and adult mice. To accomplish this goal the first two exons of the mouse Mrad9 gene in ES cells were surrounded by loxP sites, using gene targeting techniques, and subsequently deleted by expressing Cre, a site specific recombinase specific for loxP sites. These cells were then used to make heterozygous Mrad9 knockout mice. Gamma-ray survival curves for heterozygous knockout ES cells indicate that the absence of one functional copy of Mrad9 has no effect upon survival. Attempts to create homozygous Mrad9 deleted ES cells or whole mice have been unsuccessful thus far, indicating that at least one functional copy of the gene is needed for viability. Attempts are being made to circumvent the lethality of the homozygous mutation, for further studies to assess the role of Mrad9 in radioresistance-promoting mechanisms.

KEYWORDS: rad9, knockout mouse, radioresistance, cell cycle checkpoint