Due to their larger size and higher intelligence rats are superior to mice for many neuroscience fields, but for historical reasons including challenges with genome editing they have often been overlooked in preference to the mouse. However, programmable nuclease technologies such as zinc finger nucleases (ZFNs) and CRISPR/Cas9 system have made engineering rat models much easier, and in this manner it is now possible to introduce the Cre-LoxP system into the rat.
Using gene editing we have created a suite of rat models for optogenetics research, consisting of various neuron-specific Cre drivers, Cre activity-dependent fluorescent reporter and opsin-expression rat lines. You can find our complete characterization of these rats in our recent publication in PLOS ONE, but below we detail some of the analysis that demonstrates that the Cre expression in these Cre driver rats faithfully recapitulates that of the respective endogenous gene.
Rosa Tdtomato Rats Faithfully Report Cre Activity
Rosa TdTomato reporter rat (Rosa Tom) expresses Cre in Th positive neurons when mated with either Th-cre (B-C’’) or DAT-Cre (H-H’’), but not wild type(A-A’’), rats. In addition, it also demonstrated Cre activity in some Th negative neurons in the brain when mated with Th-cre (D-F), probably reflecting the transcription/activity of Th/Cre in the history of development since the labeling is indelible at genomic DNA level and no Th negative neurons are labeled by Rosa Tom when mated with DAT-Cre rats (G).
For details on the analysis of Th-cre and DAT-cre rats, please see Liu et al, PLOS One: 11(2): e0149379
Cre-Dependent Expression of eNpHR3.0-Tom
Rosa eNpHRe3.0-Tom shows expected expression of eNpHR3.0-Tdtomato fusion protein in Th positive neurons when mated with DAT-Cre rats.
SLC32A1-Cre Labels Correct Populations of Cells
A combination of Slc32A1 in situ hybridization and Tdtomato antibody staining confirms Cre activity-dependent Tdtomato labeling in Slc32A1 positive neurons in the brain (A-B’’).
Similarly, Tdtomato exhibits similar labeling pattern in SLC32A1-cre retina (D and E) as Slc32A1 antibody in wild type (F and G). Note that the signal labeled with stars in D, E and G is auto-fluorescence background.
In both Cre lines, Cre expression was detected in expected neurons. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats (read more here).
In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins.
The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson’s disease.
Find out more about: