DEVELOPMENT OF CONGENIC STRAINS/IDENTIFICATION OF THE GENE UNDERLYING LORE2

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The goals of this component are to identify the mouse gene underlying Lore2, a QTL for alcohol sensitivity located on mouse chromosome 2, and to provide congenic mouse strains for the use of other components of the University of Colorado Alcohol Research Center (CU ARC). The focus of our work is the ISS and ILS strains of mice which differ markedly in the length of loss of righting reflex (LORR) after ethanol administration. This differential LORR has been shown to be associated with several distinct chromosomal regions (QTLs), each of which explains part of the difference. Lore 2 explains about 14.2 % of the genetic variance in sleep time (a 25 minute difference between the inbreds). This QTL has been detected has been detected both in RIs (p less than 0.001) and in F2's (lodmax=6.6) and has been confirmed using directed genome selection methodologies. Lore2 is located near 85 cM of chromosome 2 (l-lod-support interval is from 78-95 cM. Component 3 will simultaneously carry out mouse breeding efforts that will narrow the Lore2 interval to less than 1 cM (Dr. Johnson) and gene identification strategies that will candidate genes in the interval for sequence differences (Dr. Sikela). We will also interact with other ARC investigators to establish functional confirmation of the gene(s) identified by this component and provide congenic strains for the other components. The mouse genetic component will perform two different functions. First, a series of congenics will be constructed carrying each short-sleep QTL on the ILS background (the complementary congenics, L alleles on an ISS background, are being developed by an RO1). Second, the Lore2 QTL will be localized to 1cM or less using markers spanning the 78-95 cM interval on chromosome 2. Both general aims use speed congenic approaches to backcrosses of ISS to ILS and the reciprocal together with typing of SSLP markers. Three rounds of backcrosses will be performed using the assistance of a speed congenic analysis followed by assessment of phenotype if mice in the fourth round and subsequent completion of seven more rounds of backcrosses. Mice will be assessed for recombination events in this interval on chromosome 2 to localize Lore2 to approximately 1cM at better than a 50% probability. For gene identification, the primary strategy will be to utilize the rapidly emerging mouse and human genome resource relating to ESTs/genes and gene maps. Through the use of these resources, mouse cDNAs that lie in the Lore2 interval will be identified and used for PCR-based direct sequence comparisons between ILS and ISS mice. Once sequence differences are obtained they will be tested by appropriate functional assays and other confirmatory methods though collaborations with other ARC laboratories. The human counterpart of the resulting Lore2 gene will be a candidate for contributing to genetic predisposition to alcoholism. (For those unfamiliar with the specialized genetics terminology, a lexicon has been provided as Table 1.)