Lead Inventor:
Franklin David Costantini, PhD
Less Difficulty in Altering a Mouse Genome
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 includes useful sequences for creating a transgenic mouse, and has been designed for use with the companion plasmid pROSA26PA for targeting 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.
Creating a Transgenic Mouse with pBigT Plasmid The pBigT plasmid is used in creating transgenic mice. The plasmid contains a loxP-flanked cassette with a PGK-neo selectable marker and a tpA transcriptional stop sequence. The plasmid pBigT consists of the adenovirus splice acceptor sequence followed by a loxP site, neo expression cassette, strong transcriptional stop sequence (triple SV40 polyadenylation sequence), another loxP site in the same orientation as the first, a multiple cloning site (MCS), and the bovine growth hormone polyadenylation sequence. A PacI site was included 5' to the SA, and an AscI site 3' to the bpA. These two enzymes are rare eight base pair cutters and result in sticky ends upon digestion and can be used to excise the entire construct, for insertion into the plasmid with the ROSA26 genomic arms. The XbaI site used for insertion into the ROSA26 genomic locus was replaced by a linker (PacI, SwaI, AscI), so that it could be digested with PacI and AscI, and receive the BigT sequence.
Applications:
• pBigT is a targeting vector used for making transgenic mice. The plasmid contains a loxP-flanked cassette with a PGK-neo selectable marker and a tpA transcriptional stop sequence.
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
b-gal in living tissue.
Patent Status: Copyright
Licensing Status: Copyright / Material available for Express Licensing
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.
