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Selected Publication:

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Publication type: Doctoral Thesis

Year: 2011

Author(s): Kautschitsch, Susanna

Title: Novel Approaches to Gene Targeting.

Other title: Neue Gene Targeting Anwendungen

Source: Dissertation, Vet. Med. Univ. Wien, pp. 129.


Advisor(s):

Rülicke Thomas

Reviewer(s):
Müller Mathias

Vetmed Research Units:
Institute of Laboratory Animal Science


Graduation date: 08.06.11


Abstract:
In order to elucidate gene function and regulation, transgenic mouse models have become indispensable to genetic research. In particular, Gene Targeting, utilizing Homologous Recombination (HR) in cultured pluripotent embryonic stem (ES) cells, has become an integral tool to study specific gene function and regulation. However, highly sophisticated microinjection techniques are also still crucial methods for generating transgenic animal models. To overcome the current technical and biological limitations in gene targeting, two innovative approaches were developed in this work. One approach deals with targeted clone identification in gene targeting experiments by visualizing targeting events utilizing the presence of fluorescence genes. Therefore, we derived a double fluorescent ES cell line from the Cre reporter Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J strain which expresses membrane-targeted tdTomato (mT) and upon Cre recombination, membrane-targeted EGFP (mG) on the Rosa26 locus. Animals were bred to achieve the desired double fluorescence expression and ES cells were derived, expressing mT and mG simultaneously from two corresponding alleles. By targeting the Rosa26 locus, the respective targeting construct recombines against one of the fluorescence genes, resulting in the loss of one fluorescence gene or signal, respectively. In two targeting experiments on two 96-well plates each, we were able to identify six clones, exhibiting the loss of the mG locus. To date, efficient gene targeting is restricted to ES cells and involves laborious cell culture and screening procedures for targeted clones. To circumvent this extensive technology, a transgenic oocyte donor model (Zp3-Rad51) was designed, generated, genotyped and phenotyped. This murine model overexpresses Rad51, a key protein in the process of HR, under the transcriptional control of the murine Zp3 promoter, being active during oogenesis. Conventional pronuclear microinjection into zygotes resulted in five transgenic founder lines. RT-qPCR analysis revealed that one line is expressing transgenic Rad51 mRNA in oocytes. To assess the potential of this mouse model to efficiently induce HR in oocytes, this line will be applied to microinjection experiments with gene targeting constructs. Successful gene targeting events in oocytes or early embryos would bypass embryonic stem (ES) cell technology and therefore result in faster and more efficient gene targeting experiments. Both approaches aim to solve fundamental limitations in transgenic research and will potentially change the way of approaching transgenic animal model design.


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