title: Combining fibroblast isolation with lentiviral gene transfer to validate transgene expression in mice following pronucleus injection.
abstract: The binary tetracycline-based expression system in transgenic mice relies on the expression of the tetracycline transactivator (tTA or rtTA) in a particular cell type together with a transcription unit encoding the gene of interest under a tetracycline or doxycycline-responsive promoter. Transgenic mice containing this transcription unit are produced via pronucleus injection. As the chromosomal integration site of the injected DNA influences transgene expression, several founder lines have to be crossed with (r)tTA-expressing mice to find a line showing low background and high transgene expression following doxycycline stimulation. Here, we describe a method to analyze primary fibroblasts derived from the founder lines to quickly test transgene expression and inducibility. Fibroblasts isolated from a small piece of mouse ear were infected with a recombinant lentivirus expressing rtTA. Transgene expression was verified by both RT-PCR and western blot, following stimulation with doxycycline. Transgene expression could easily be detected on the RNA and protein levels in primary fibroblasts derived from transgenic founder lines. An enzymatic function of the transgene was not required for the identification of transgene expression. Thus, the method allows a quick and easy discrimination of transgenic founder lines according to transgene expression and inducibility.
authors: Department of Medical Biochemistry and Molecular Biology, Saarland University, 66421, Homburg, Germany.
authors: Oliver G
authors: OG
authors: Rössler
authors: Department of Medical Biochemistry and Molecular Biology, Saarland University, 66421, Homburg, Germany.
authors: Andrea
authors: A
authors: Lesch
authors: Department of Medical Biochemistry and Molecular Biology, Saarland University, 66421, Homburg, Germany. gerald.thiel@uks.eu.
authors: Gerald
authors: 0000-0001-9163-8242
authors: G
authors: Thiel
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000235:genetics*; D004318:Doxycycline / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation; D019076:Transgenes / Q000235:genetics*
chemical_list: D004318:Doxycycline
keywords: Elk-1; Lentivirus; Tet expression system; Transgenic mice