Plasmid for mouse ROSA26 locus for use with plasmid pBigT

Lead Inventor: Franklin David Costantini, PhD Creating a Transgenic Mouse In biomedical research, a very common technique is to alter a mouse's genome to induce or repress expression of a gene of interest. Although this technology has been around for many years, the actual laboratory work required to produce such a mouse is time-consuming and prone to failure. This technology consists of a plasmid that has been optimally designed to insert any DNA sequence of choice into the mouse genome at the ROSA26 locus. This locus is important because it has a high endogenous expression level throughout development; thus, the inserted sequence will be expressed globally, or can be restricted in time and/or space by using a second 'driver' mouse line. Using pROSA26-PA in Conjunction with pBigT to Alter a Mouse Genome • The technology consists of the generic targeting vector, called pROSA26-PA, which can be used in conjunction with pBigT (see IR #2446) to easily target the ROSA locus. • A scientist wishing to create a gene insertion first assembles the desired sequence, subclones it into the pBigT vector, and then moves a larger fragment from pBigT into the pROSA26-PA vector. • After this step, the final targeting sequence is extracted and inserted into the mouse genome (e.g., by embryonic stem cell transformation). • This technology makes targeting of the ROSA26 locus straightforward by incorporating several features: o Rare-cutting restriction enzyme sites AscI and PacI allow for 'sticky-end' ligation, obviating the need for more time-consuming types of ligation. o The companion pBigT plasmid includes useful ancillary sequences such as PGK-neoPGK-pA and loxP sites, for use with Cre recombinase. • This technology was amply demonstrated in the creation of Cre reporter strains using gene-encoded fluorescent tags Applications: • These targeting constructs are used to create mice suitable for cell lineage tracing experiments as well as for monitoring the expression of Cre transgenes using fluorescent protein marker expression. Advantages: • The green fluorescent protein (GFP) and variants such as EYFP and ECFP offer an advantage over lacZ as a reporter, in that they can be easily visualized without recourse to the vital substrates required to visualize β-gal in living tissue. Patent Status: Copyright Licensing Status: Copyright / Material available for Express Licensing Plasmid for mouse ROSA26 locus for use with plasmid pBigT Publications: Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. Srinivas, S et al. (2001). BMC Dev Biol. 1:4.