Journal: bioRxiv
Article Title: Generation and characterization of a tamoxifen-inducible, Cre driver rat for transgene expression in microglia
doi: 10.1101/2025.07.17.665447
Figure Lengend Snippet: (A) The DIO-mCherry rat uses human EF1α promoter to drive expression of mCherry fluorescent protein along with the woodchuck post transcriptional regulatory element (WPRE) and the human growth hormone polyadenylation signal (hGHpA). (A) The primer positions for a PCR genotyping assay spanning the 5’ junction of the mCherry cassette is noted by the black arrows and the positions of the primers (blue) and probe (green arrow) for a qPCR-based genotyping assay are also noted. ( B ) Copy number for WT, hemizygous (Hem), and homozygous (Hom) animals. The ratio is transgene copy number to the genomic gene (Ggt1) in genomic DNA samples isolated from the pups of two separate Hem × Hem crosses. The individual ratios of templates (DIO-mCherry/Ggt1) fall into three quanta (0, 0.5, and 1.0). (C) Amplicons (640 bp) produced by the 5’ junction PCR identify wild type (no bands) or carriers hemizygous and homozygous. (D) Images from the striatum of hemizygous DIO-mCherry rats injected with an AAV virus expressing Cre recombinase after two weeks. Low magnification (top row; scale bar: 500 μm) and higher magnification (bottom row; scale bar: 50 μm). Cre immunoreactivity (white) and mCherry epifluorescence (red) colocalize in both sets of images. Overlap Coefficient (OC)=0.90.
Article Snippet: The coding region for CreERT2 was amplified from MSCV CreERT2 puro (a gift from Tyler Jacks; Addgene; #22776) and inserted into an AAV packaging plasmid flanked by the EF1α promoter and the poly-adenylation signal from bovine growth hormone using ligation-independent cloning (In-Fusion, Clontech).
Techniques: Expressing, Genotyping Assay, Isolation, Produced, Injection, Virus