Making a transgenic mouse with α 2A adrenoceptor fused with GFP

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This project is to make transgenic mouse with alpha2A adrenergic receptor (alpha2A AR) fused with green fluorescent protein (GFP). The targeting vector was made by modifying the HA tagged vector (30 bp was added around the initiation codon of the alpha2A AR) which had been gifted from the Department of Pharmacology, the Vanderbilt University. The targeting vector was transfected by electropolation into the ES cell derived from mouse strain 129 SvEv. After positive-negative selection by G418, PCR screening showed 10 positive colonies. The Southern blotting revealed 3 positive clones. Two clones was micro-injected into blastcyte of C57BL/6 mouse. We got 3 female and 2 male chimerae. Mating of Fl hetero-mouses resulted in 41 hetero type (20 males and 21 females) with no homozygotes. Immunohistochemistry revealed that GFP was expressed in locus coeruleus in the heterozygotes, though the fluorescent intensity was not so strong. Therefore, we are trying to get homo-type mouse by using the other ES cell lines.Although we have not yet published about this study because homo-type mice have not yet got, no one has ever been able to produce this type of transgenic mouse. We are going to perform the physiological analysis as soon as getting homo-type mouse, and to consider the collaboration with other institutes. Also, we are going to apply the multiphoton excitation-evoked chromophore-assisted laser inactivation to antagonize the anesthesia by dexmedetomidine (alpha2 agonist) in this transgenic mouse.