Vector and Method to Visually Genotype Transgenic Animals

Technology #12612

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David R. Borchelt
Hilda H. Brown
Susan E. Fromholt
Managed By
April Kilburn
Assistant Director 352-392-8929

Gene Vector that Causes Transgenic Animals to Lightly Fluoresce for Visual Identification

Scientists at the University of Florida have developed a wide variety of monoclonal and polyclonal antibodies, cell lines, vectors and biological materials that can be used in other research projects. Anyone interested in accessing these materials via a license from the University of Florida should contact our office to make arrangements. A reporter transgene encoding enhanced green fluorescent protein (eGFP) has been designed to be co-injected with various transgene constructs. The eGFP reporter was constructed by inserting the cDNA into a vector harboring a keratin-14 (K14) promoter to drive expression of eGFP in skin. The K14 vector was created by removing the promoter element of the MoPrP.Xho vector and replacing it with a 2 kb fragment of the human keratin-14 gene. Previous studies have shown that co-injection into single cell embryos of two independent transgenes results in co-integration of the transgenes at a single locus. In our experience, the two transgenes are so closely integrated that recombination to segregate the genes virtually never occurs. The transgene construct to be tested is co-injected with the K14-eGFP construct. Using goggles fitted with the appropriate filters to visualize eGFP expression, we could reliably distinguish transgenic mice from nontransgenic mice. The expression of eGFP persists into adulthood and is most visible in ears, paws and tail skin. Mice harboring the K14-eGFP transgene alone appeared normal throughout their lifespan (more than 530 days).

The Keratin14-eGFP expression vector was made by amplifying the Keratin14 promoter using the following primers: 5’-CCGGATCCGCGGCCGCCTCCGGAGCTTCTATTCC-3’ and 5’-CCGGATCCTAAATTGGAAAGGGATGCGAGTGC-3’. The 2 kb amplicon was digested with BamHI (underlined sequence) and ligated into the PrP-Tet promoter vector, which is a variation of the MoPrP.Xho vector in which the PrP promoter elements have been replaced by the tetracycline operator. The tetracycline operator is flanked by BamHI restriction sites, allowing for easy excision and replacement. The eGFP cDNA was excised from its vector (Clontech #6086-1, Accession #U55761) and ligated into the XhoI cloning site of the new K14.PrP.Xho vector.