Tissue-Specific Regulation of Oncogene Expression Using Cre-Inducible ROSA26 Knock-In Transgenic Mice

Abstract

Cre-inducible mouse models are often utilized for the spatial and temporal expression of oncogenes. With the wide number of Cre recombinase lines available, inducible transgenesis represents a tractable approach to achieve discrete oncogene expression. Here, we describe a protocol for targeting Cre-inducible genes to the ubiquitously expressed ROSA26 locus. Gene targeting provides several advantages over standard transgenic techniques, including a known site of integration and previously characterized pattern of expression. Historically, an inherent instability of ROSA26 targeting vectors has hampered the efficiency of developing ROSA26 knock-in lines. In this protocol, we provide individual steps for utilizing Gateway recombination for cloning as well as detailed instructions for screening targeted ES cell clones. By following this protocol, one can achieve germline transmission of a ROSA26 knock-in line within several months.

Keywords: Cre-inducible; Gateway; In-Fusion; JM8A3 ES cells; ROSA26; Southern blotting; oncogene.

Figure 1

Schematic of steps required for inserting a cDNA of interest into pRosa26-DEST. A cDNA is amplified using PCR to add In-Fusion (IF) adapter sequences. The In-Fusion reaction is then performed between the PCR product and linearized Entry vector to obtain a Gateway-ready Entry vector containing the cDNA. This vector is then combined with pRosa26-DEST in a LR reaction to obtain to targeting vector.

Figure 2

Targeting the ROSA26 locus for inducible expression of a gene of interest. A) A cassette containing Neomycin as a selectable marker driven by the PGK promoter, followed by 4X poly A (pA) signals is flanked by loxP sequences (black triangles), and placed downstream of a splice acceptor (sa) site in the ROSA26 locus. A gene of interest may be followed by an intervening sequence and a reporter gene such as green fluorescent protein. B) All cells that will grow in neomycin are screened by PCR to determine if they contain the cassette. The Forward (F) primer in the 5′ homology arm also binds to WT ROSA sequence, but the Reverse (R) primer is downstream in unique sequence (does not bind to WT ROSA) (black arrows in A). C) Positive ES cell clones detected by PCR are expanded, and screened by Southern blot using a unique sequence probe from the 5′ end (black line), and from the 3′ end (black line). Restriction enzyme X and Y cut within the gene of interest to produce a smaller product than the wild type. The appropriate size predicts correctly targeted clones. These probes can be used to confirm correctly rearranged alleles after deletion of the floxed STOP cassette with a Cre.

Figure 3

Representative images of ROSA26 knock-in chimeras and progeny generated using JM8A3 ES cells. A) An ~50% coat color contribution chimera. B) An ~90% coat color contribution chimera. C) A representative white-eyed (white arrow), C57BL/6-Tyr^c-Brd albino blastocyst-derived pup, and a representative black-eyed (black arrow), C57BL/6N JM8A3-derived pup.