Beth Bennett, Ph.D.
Institute for Behavioral Genetics
Campus Box 447
University of Colorado at Boulder
Boulder, CO 80309-0447
sensitivity in ILSxISS derived mice
The Long Sleep and Short Sleep mice have been selected for sensitivity (LS) and lack of sensitivity (SS) to alcohol. These mice, and their various derivatives (ILS, ISS, and RIs), are the most studied animal model of the effects of ethanol on the nervous system.
We have been pursuing the genetic basis of this differential sensitivity. We used a two-stage strategy: first mapping quantitative trait loci (QTLs) for ethanol sensitivity in the LSxSS RI series1 (consisting of 25 strains) then mapped the same trait in 1063 ILSxISS F22. We confirmed three of the QTLs originally identified in the RIs and identified 3 additional QTLs in the F2.
We are now generating congenic lines in which each of the major QTLs has been moved onto the opposite genetic background3. For example, a single ILS QTL has been moved onto an ISS background. The reciprocal lines have also been created. We used a combination of marker-assisted selection and phenotyping to create these congenic mice. We are currently at generation 8-10 of the backcrossing. Standard congenic methodology requires at least 10 generations of backcrossing to reduce unlinked donor background to below 0.2%.
The congenic lines in which an ILS QTL was moved onto the ISS background have been tested for ethanol sensitivity since the 4th generation of backcrossing. These results were important in that we were able to identify lines in which the QTL had not been captured, eliminate them, and concentrate on positive lines. Only the ILS QTLs are amenable to this type of analysis as the added effect due to a single gene is sufficiently large (approximately 10 minutes) as to allow its detection on the ISS background (approximately 20 minutes of sleep time) in a relatively small sample size. At the end of ten generations of backcrossing, mice heterozygous for the one type of QTL (e.g. the ILS) will be intercrossed to generate true congenics, that is, lines of mice homozygous for that QTL on the opposite background. These mice will be valuable for validating QTLs for ethanol sensitivity as well as for testing hypotheses regarding the genetic basis for effects of other drugs.
We are also generating lines of mice carrying chromosomes recombinant in the QTL regions. These lines will enable us to narrow the region surrounding the QTL, which is currently over 20 cM in most cases. Recombination events in the congenic mice allow us to select chromosomes in which this interval has been reduced. We then backcross these mice to ISS and test the progeny. If recombinant progeny maintain a higher sleep time than non-recombinant offspring then we can conclude that the QTL is contained within the smaller interval.
1. Markel PD, Fulker DW, Bennett B, Corley RP, DeFries JC, Erwin VG, Johnson TE: Quantitative trait loci for ethanol sensitivity in the LSXSS recombinant inbred strains: Interval-mapping. Behav. Genet. 26: 447-458, 1996
2. Markel PD, Bennett B, Beeson M, Gordon L, Johnson TE: Confirmation of quantitative trait loci for ethanol sensitivity in long-sleep and short-sleep mice. Gen Res 7:92-99, 1997
3. Bennett, B, Johnson, TE: Development of congenics for hypnotic sensitivity. Mamm Genome 9, 969-974, 1998