Quantitative Trait Loci (QTL) are chromosome sites containing alleles (genes) that influence a continuously distributed (quantitative) trait. Recently developed molecular and statistical methods utilizing PCR-based and RFLP marker loci now make it possible to detect and genetically map several QTL determining the intensity of withdrawal from drugs of abuse. Using this approach, we have very recently gathered evidence strongly suggesting the chromosome location of a QTL affecting ethanol withdrawal intensity. Following initial screening for candidate QTL using the BXD Recombinant Inbred strain panel (BXD RIs), individual F2 mice from a cross between C57BL/6J and DBA/2J strains were tested. These two parental strains differ markedly in ethanol withdrawal intensity after the same ethanol exposure. Phenotype (i.e., ethanol withdrawal severity) of individual F2 animals was related to their genotype, which was determined by PCR amplification of polymorphic markers flanking the putative QTL. We were able to demonstrate that phenotypic scores were highly related to genotype for the D2Mit9 locus, located 38 cM from the centromere on mouse chromosome 2, in a region highly syntenic (homologous) with a region of human chromosome 2q. Using similar techniques, we propose to identify and map several QTL, each accounting for 20% or more of the genetic variance for withdrawal intensity following diazepam, pentobarbital, and morphine. Selective breeding for the QTL will be used to isolate the high and low predisposing alleles in genotypic selected lines. From these lines, the most promising will be developed into congenic inbred strains through repeated backcrossing with one of the parental inbred strains. These strains will possess a small chromosome segment (1-2% of the genome) containing a known QTL from the C57BL/6J strain superimposed on a genetic background that is 98-99% from the DBA/2J strain, and vice-versa. Because of the near elimination of genetic """"""""noise"""""""" at irrelevant loci, these lines will be valuable in the identification of a QTL(candidate gene) playing a major role in withdrawal, and should facilitate investigations of the cellular mechanisms responsible for adaptation to drugs of abuse. We will also determine the influence of each QTL on withdrawal intensity associated with ethanol, pentobarbital, diazepam, nitrous oxide and morphine by comparing the pairs of oppositely-selected genotypic selected lines and congenic strains. These studies should help to identify specific candidate genes affecting drug withdrawal. Furthermore, preliminary data collected during the initial grant period suggests strongly that some QTL we identify will influence withdrawal from multiple drugs of abuse. For example, the QTL near D2Mit9 also affects severity of withdrawal from nitrous oxide. Any QTL affecting multiple drugs of abuse will receive priority in our investigations.
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