Author/Editor		Kotnik, Katarina; Mutzel, Rupert; Bader, Michael
Title		Gene knockdown in transgenic rats by shRNA technology
Type		monografija
Place		Berlin
Publisher		[K. Kotnik]
Publication year		2008
Volume		str. 125 str.
Language		eng
Abstract		The lack of germline-competent ES celIs makes gene inactivation throughhomologous recombination impossible in the rat. The main goal of this thesis was tosolve this problem by establishing a new gene down regulation technology usingshRNA, a natural phenomenon and a powerful tool for gene knockdown inmammalian celIs and mice. Three DNA constructs harbouring hairpin cassettesagainst the EGFP gene and controlled by the U6 promoter were generated. Afterstandard pronuclear microinjection into zygotes of EGFP transgenic rats more thanthree hundred rats were bom. Surprisingly, only one animal was found to carry theconstruct integrated into the genome. No GFP silencing was observed in thistransgenic founder nor was any shRNA expression detectable. Even the use of BACconstructs based on the mouse Rosa26 locus to achieve single transgene integrationdid not result in successful generation of shRNA transgenic rats. Suspecting embryo-toxicity by abundant production of shRNA molecules an in vitrostudy was carried out. By assaying embryonic development and survival rate of ratand mouse embryos after introduction of a hairpin construct it was shown that shRNAexpression does not disturb embryonic development at early preimplantation stages. Consequently, the problem may appear later during pregnancy. Since pronuclear microinjection of ubiquitously and permanently active shRNAconstructs does not work in the rat as shown in this thesis, a novel strategy to achieveconditional gene knockdown was tested. For this purpose, a tetracycline activatablesystem was used to control the expression of shRNA against the insulin receptor(InsR), resulting in an inducible model of insulin resistance. Consequently, aninducible and chronic model of type II diabetes including several hallmarks of thisdisease was established. In addition, anovel inducible expression system for shRNA was developed using aligand-dependent chimeric transcription factor. As demonstrated in celI culture, amutated TATA-binding protein (mTBP) activates a U6 promoter with acorresponding TATA box mutation. The fusion of a mutated ligand binding domain of an estrogen receptor to the mTBP leads to a chimeric protein. The translocation of the fusion protein into nucleus should depend on the presence of tamoxifen. However, this ligand-inducibility of the mTBP could not be achieved in this thesis. In conclusion, with the use of the doxycycline inducible shRNA expression system, time and/or dose dependent gene in hibition in rats becomes possible. This flexible gene manipulation strategy in the preferred animal model for physiopathological studies may provide a powerful tool for the understanding of gene function.
Keywords		genetika shRNA regeneracija gena inzulin diebetes melitus