Abstracts

BACTERIAL ARTIFICIAL CHROMOSOME (BAC) TRANSGENESIS CONFIRMS EFFECT OF A QUANTITATIVE TRAIT LOCUS FOR SEIZURE SUSCEPTIBILITY IN MICE: FURTHER EVIDENCE THAT CODING VARIATION IN KCNJ10 INFLUENCES SEIZURE THRESHOLD

Abstract number : 3.026
Submission category :
Year : 2005
Submission ID : 5832
Source : www.aesnet.org
Presentation date : 12/3/2005 12:00:00 AM
Published date : Dec 2, 2005, 06:00 AM

Authors :
1Russell J. Buono, 2Gregory T. Golden, 3John P. Dahl, 2George G. Smith, 3Candice L. Schwebel, 2James F. Martin, 3Ross MacDonald, 3Falk W. Lohoff, 3Wade H. B

Quantitative trait loci (QTL) mapping studies from our lab identified a 6.1 Mb segment of distal chromosome 1 that contains a gene (or genes) having a major influence on the difference in seizure susceptibility between C57BL/6 (B6) and DBA/2 (D2) mice. A random insertion gene transfer strategy involving a BAC DNA construct that contained four tandem B6-derived candidate genes from the critical interval (Kcnj9, Kcnj10, Igsf8 and Atp1a2) was used to test the hypothesis that a strain-specific variation in Kcnj10 is responsible for the QTL effect. Fertilized oocytes from the seizure-sensitive congenic strain B6.D2-Mtv7a/Ty-27d were injected with RPCI-23 157J4 BAC DNA and three independent founder lines of BAC-transgenic (TG) mice were generated. TG and wildtype (WT) progeny were phenotyped by measuring maxiaml electroshock seizure threshold (MEST). Allele-specific RT-PCR analysis documented brain expression of BAC-containing candidate genes. Quantitative RT-PCR was used to measure brain mRNA levels and western blots were used to quantify brain protein levels of the BAC derived genes in TG and WT mice. Quantification of seizure susceptibility documented significantly higher maximal electroshock seizure thresholds in TG mice compared to WT littermates. Relative levels of Kcnj10, Kcnj9 and Atp1a2 mRNA were 1.0-2.0 fold higher in TG mice compared to WT. Western analysis revealed close correspondence between regional brain levels of Kcnj9 and Kcnj10 encoded proteins and their respective mRNAs although there was regional variability. Taken together, results suggest that one or more of the BAC derived genes contain a polymorphism that is responsible for the previously documented chromosome 1 seizure QTL ([italic]Szs1[/italic]). Given the lack of major expression differences noted for any of the BAC derived genes studied and the 100% identity of amino acid sequence between B6 and D2 mice for Kcnj9, Igsf8 and Atp1a2, the non-synonymous (Thr262Ser) SNP in Kcnj10 remains the variation that is most likely to define the seizure related QTL [italic]Szs1[/italic]. (Supported by NIH Grants R01NS40396 to RJB and R01NS40554 to TNF, University of Cincinnati Department of Neurology, University of Pennsylvania Center for Neurobiology and Behavior.)