GABA_A receptors mediate the major inhibitory synaptic transmission in the central nervous system, and form a number of binding sites for clinically important drugs such as benzodiazepines. Alterations in GABAergic neurotransmission have been implicated in the pathogenetics of a number of neuropsychiatric diseases and disorders. GABA_A receptor αl subunit is the most widely expressed in the brain among at least 17 known subunits. This single fact implies that αl subunit may play important roles in the brain. Although it has been suggested by in vitro studies that αl may involve in functions such as the benzodiazepine binding, the function of this subunit has not been addressed directly in vivo....[ Read more ]

GABA_A receptors mediate the major inhibitory synaptic transmission in the central nervous system, and form a number of binding sites for clinically important drugs such as benzodiazepines. Alterations in GABAergic neurotransmission have been implicated in the pathogenetics of a number of neuropsychiatric diseases and disorders. GABA_A receptor αl subunit is the most widely expressed in the brain among at least 17 known subunits. This single fact implies that αl subunit may play important roles in the brain. Although it has been suggested by in vitro studies that αl may involve in functions such as the benzodiazepine binding, the function of this subunit has not been addressed directly in vivo.

Gene targeting is by far the most advanced technique for studies of gene functions in vivo. The present thesis work aimed at the construction of a replacement type of vector for a targeted disruption of the GABA_A receptor αl subunit in mice. The success of this construction will facilitate the ablation of αl subunit gene and provide a mouse model for the in vivo studies on GABA_A receptor αl subunit.

For the construction of the targeting vector, a 13-kb DNA fragment containing the first two exons of mouse GABA_A receptor al subunit was obtained from screening of a λ2001 based 129/Sv mouse genomic library. After characterizations by restriction digestions and DNA sequencing, a 6 kb fragment corresponding to part of the 5' non-coding sequence of the gene and a 1.8 kb fragment harboring Exon 2 of the gene were inserted into the targeting vector flanking a neomycin resistant gene (neo^R). A copy of thymidine kinase (PGK-TK) gene was incorporated into the vector adjacent to the 1.8 kb fragment. The vector was linearized and electroporated inito cultured Rl mouse embryonic stem (ES) cells, which were maintained at the undifferentiated state by the inclusion of leukaemia inhibitory factor (LIF) into the culture media. ES cells carrying neo but not PGK-TK were selected with medium containing G418 and Ganciclovir. One hundred and thirty-eight drug-survival clones were obtained. Strategies for screening from these clones of targeted ES cells by PCR and Southern hybridization were formed. These will allow the identification of ES cells with the Exon l of αl subunit gene being replaced by neo gene. Therefore, mice generated from these cell lines will have ablated GABA_A receptor αl subunit.