ABSTRACT
Thanks to many advances in genetic manipulation, mouse models have become very powerful in their ability to interrogate biological processes. In order to precisely target expression of a gene of interest to particular cell types, intersectional genetic approaches using two promoter/enhancers unique to a cell type are ideal. Within these methodologies, variants that add temporal control of gene expression are the most powerful. We describe the development, validation and application of an intersectional approach that involves three transgenes, requiring the intersection of two promoter/enhancers to target gene expression to precise cell types. Furthermore, the approach uses available lines expressing tTA/rTA to control the timing of gene expression based on whether doxycycline is absent or present, respectively. We also show that the approach can be extended to other animal models, using chicken embryos. We generated three mouse lines targeted at the Tigre (Igs7) locus with TRE-loxP-tdTomato-loxP upstream of three genes (p21, DTA and Ctgf), and combined them with Cre and tTA/rtTA lines that target expression to the cerebellum and limbs. Our tools will facilitate unraveling biological questions in multiple fields and organisms.
Footnotes
Author contributions
Conceptualization: A.R.-D.; Methodology: A.R.-D., A.L.J.; Formal analysis: N.S.B., A.S., A.R.-D.; Investigation: E.A., N.S.B., X.Q., A.S., L.M., D.S., A.R.-D.; Writing - original draft: E.A., A.R.-D.; Writing - review & editing: N.S.B., A.L.J., A.R.-D.; Supervision: A.L.J., A.R.-D.; Project administration: A.L.J., A.R.-D.; Funding acquisition: A.L.J., A.R.-D.
Funding
This work was supported by grants from the Human Frontier Science Program (LT000521/2012-L and CDA00021/2019) and the Charles H. Revson Foundation (grant number 15-34) to A.R-D.; Monash University Graduate Scholarship, Monash University International Tuition Scholarship and Islamic Development Bank (600037455) funds to E.A.; Postdoctoral fellowships from New York State Stem Cell Science (C32599GG) and the National Institute of Neurological Disorders and Stroke (K99 NS112605-01) to N.S.B.; the National Institutes of Health (R21 HD083860, R37 MH085726 and R01 NS092096) to A.L.J. and a National Cancer Institute Cancer Center Support Grant (P30 CA008748-48) to Memorial Sloan Kettering Cancer Center Deposited in PMC for release after 12 months.