Publication Date
1-28-2022
Journal
BMC Biology
DOI
10.1186/s12915-022-01227-0
PMID
35086530
PMCID
PMC8796356
PubMedCentral® Posted Date
1-28-2022
PubMedCentral® Full Text Version
Post-print
Published Open-Access
yes
Keywords
Animals, Calcium, Clustered Regularly Interspaced Short Palindromic Repeats, Female, Integrases, Mice, Mice, Transgenic, Neurons, Recombinases, Intersectional genetics, Gene targeting, CRISPR/Cas9, Cre, Flp, Dre, DREADDs, Fluorescent reporter
Abstract
BACKGROUND: The functional understanding of genetic interaction networks and cellular mechanisms governing health and disease requires the dissection, and multifaceted study, of discrete cell subtypes in developing and adult animal models. Recombinase-driven expression of transgenic effector alleles represents a significant and powerful approach to delineate cell populations for functional, molecular, and anatomical studies. In addition to single recombinase systems, the expression of two recombinases in distinct, but partially overlapping, populations allows for more defined target expression. Although the application of this method is becoming increasingly popular, its experimental implementation has been broadly restricted to manipulations of a limited set of common alleles that are often commercially produced at great expense, with costs and technical challenges associated with production of intersectional mouse lines hindering customized approaches to many researchers. Here, we present a simplified CRISPR toolkit for rapid, inexpensive, and facile intersectional allele production.
RESULTS: Briefly, we produced 7 intersectional mouse lines using a dual recombinase system, one mouse line with a single recombinase system, and three embryonic stem (ES) cell lines that are designed to study the way functional, molecular, and anatomical features relate to each other in building circuits that underlie physiology and behavior. As a proof-of-principle, we applied three of these lines to different neuronal populations for anatomical mapping and functional in vivo investigation of respiratory control. We also generated a mouse line with a single recombinase-responsive allele that controls the expression of the calcium sensor Twitch-2B. This mouse line was applied globally to study the effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on calcium release in the ovarian follicle.
CONCLUSIONS: The lines presented here are representative examples of outcomes possible with the successful application of our genetic toolkit for the facile development of diverse, modifiable animal models. This toolkit will allow labs to create single or dual recombinase effector lines easily for any cell population or subpopulation of interest when paired with the appropriate Cre and FLP recombinase mouse lines or viral vectors. We have made our tools and derivative intersectional mouse and ES cell lines openly available for non-commercial use through publicly curated repositories for plasmid DNA, ES cells, and transgenic mouse lines.
Included in
Biochemistry, Biophysics, and Structural Biology Commons, Biology Commons, Genetics Commons, Genomics Commons, Medical Anatomy Commons, Medical Genetics Commons, Medical Molecular Biology Commons, Medical Specialties Commons
Comments
Associated Data