Different Genetic Ways for Conditional Gene Regulation in the Mouse Brain
Summary: The conditional gene expression system controlled by the tetracyclineresponsive trans-activator (tTA) in transgenic mice is an important and well characterized genetic tool. In my studies I investigated the function of the Tetregulated genes in the brain when inserted into the chromosome and when applied as extra-chromosomal elements with recombinant Adeno-Associated Virus (rAAV). First, the pool of tTA expressing mouse lines was increased by generating eight functional itTA-expressing TgThy-1.2(xx)-itTAnls transgenic mouse lines. Since for tTA expression the Thy-1.2 promoter was used, which is known to be highly integration-site dependent, lines were obtained which all differed in tTA expression level, expression pattern and ontogenetic profile in the central nervous system (CNS). These mice are a valuable tool for studies on physiological functions of different neuron populations during various developmental stages. Second, the effect of the chromosomal insertion site of Tet-responder genes on their expression was analyzed. In three different Tet-responder mouse lines, the precise integration sites and copy number of tet-responder genes were determined. No correlation between copy number of the Tet-responder gene and its expression was detected, supporting the finding that the chromosomal insertion site has strong effects on the expression of the transgene. Strong Tet-responder gene expression was observed in mice of the transgenic line SA87.5, which has three copies of a transgene inserted in the Pik3c3 gene locus. By retargeting the same position of the Pik3c3 gene locus in embryonic stem cells, only one copy of the transgene was introduced. However, due to the presence of the neo selection marker the single Tet-responsive transgene showed poor induction by tTA. After Cre-virus mediated neo removal, the expression of the Tet-responsive transgene was improved. The limitations of chromosomally inserted Tet-regulated gene elements can be overcome by using the recombinant Adeno-Associated virus (rAAV) mediated gene delivery system to introduce the different elements of the Tet-system into the CNS. It is shown that both Tet-activators and Tet-responder genes are functionally delivered by rAAV vectors, and tissue specific, homogeneous, rapid and robust expression of multiple proteins simultaneously controlled by tTA in rAAV infected CNS areas can be achieved. Moreover these viruses were used to demonstrate that the transcriptionally inactive Tet-responder genes are often epigenetically silenced during development when inserted into the host chromosome, but remain inducible when introduced extra-chromosomally after development.
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