LE-Tg(DAT-iCre)6Ottc

Background | Status & Availability | Transgene Info | Breeding | Genotyping | References | Blog/Comments/Reviews | Related rats | Acknowledgements

Background

The dopamine transporter (DAT) protein is selectively expressed in a population of dopaminergic neurons mostly concentrated in the midbrain.   Loss of DAT-expressing cells is often a marker of neurodegeneration associated with Parkinson’s disease and methamphetamine abuse.  We have generated and characterized a strain of transgenic Long Evans rats expressing iCre recombinase under a DAT promoter (DAT::iCre, line 6).  The tissue-specific expression of iCre can be used in combination with Cre-dependent transgenes to obtain selective expression of transgenes in DAT(+) neurons.

Status and Availability

This rat has been published (PMID: 30792150).
As of May 17, 2016, this strain is available as line #00758 at the RRRC.
This rat is registered at the Rat Genome Database (RGD) ID# 9588578.

Transgene Information

Figure 1:  Schematic of DAT-iCre transgenic construct.  A bacterial artificial chromosome (BAC) containing the rat DAT gene (CH230-275E16) was obtained from the Children’s Hospital Oakland Research Institute (CHORI), and recombineered to replace the start codon of DAT with cassette containing iCre (improved Cre recombinase) and the polyadenylation signal from the gene for bovine growth hormone (pOTTC173).  This BAC was injected into the pronuclei of fertilized Long Evans rat embryos by NIMH Transgenic Core, and ultimately resulted in three independent, phenotypically positive DAT::iCre lines.  This line (LE-Tg(DAT-iCre)6Ottc) has one copy of the transgene per haploid genome as determined by droplet digital PCR.

Phenotypic Characterization

Figure 2:  RNA expression patterns of DAT (red) and iCre (green) in the midbrain.   Labeling  of target mRNA was done by in situ hydridization with fluorescently-labeled RNAscope probes.  Wildtype rats show no iCre expression, whereas transgenic DAT-iCre6 rats show iCre expression only in cells that also express DAT.  The nuclei of all cells are stained with DAPI (blue).  Scale bar represents 50 microns.

Figure 3.  Midbrain expression of tyrosine hydroxylase (TH) and red to green fluorescent protein reporters of Cre activity.  Two weeks after injection of AAV-Nuc-flox(mCherry)-eGFP into the substantia nigra of a DAT-iCre6 rat brains were sections and immunostained for TH as a reliable marker for dopaminergic cells.  Transduced cells that do not express iCre are red because they only express nuclear-localized mCherry, whereas transduced (dopaminergic) cells which express iCre are green because the viral genome has recombined to delete the mCherry coding region.  The nuclei of all cells are stained with DAPI (blue).  Scale bar represent 50 microns.

Breeding Strategy

Breeding Information, click here for PDF

Genotyping Assays

Assay for the presence of DAT-iCre insert, click here for PDF

References that cite this rat

@article{Back:2019aac,

title = {Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.},

author = {Susanne Back and Julie Necarsulmer and Leslie R Whitaker and Lamarque M Coke and Pyry Koivula and Emily J Heathward and Lowella V Fortuno and Yajun Zhang and Grace C Yeh and Heather A Baldwin and Morgan D Spencer and Carlos A Mejias-Aponte and James Pickel and Alexander F Hoffman and Charles E Spivak and Carl R Lupica and Suzanne M Underhill and Susan G Amara and Andrii Domanskyi and Jenni E Anttila and Mikko Airavaara and Bruce T Hope and Kent F Hamra and Christopher T Richie and Brandon K Harvey},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30792150},

doi = {10.1016/j.neuron.2019.01.035},

issn = {1097-4199 (Electronic); 0896-6273 (Linking)},

year  = {2019},

date = {2019-04-03},

urldate = {2019-04-03},

journal = {Neuron},

volume = {102},

number = {1},

pages = {105--119},

address = {Molecular Mechanisms of Cellular Stress and Inflammation Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA.},

abstract = {Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

Blog/Comments/Reviews

There are 4 surveyed reports for the receiving and usage of Long Evans transgenic DAT iCre rats.

General Health
There are no reports of general health issues with the LE-Tg(DAT-iCre)3Ottc rats

Weight
There are no reports of weight changes with the LE-Tg(DAT-iCre)3Ottc rats
Breeding
There are no reports of breeding issues with the LE-Tg(DAT-iCre)3Ottc rats

Expression
There are no reports of expression issues with the LE-Tg(DAT-iCre)3Ottc rats

LE-Tg(DAT-iCre)6Ottc
There have been no reported issues with the Long Evans transgenic DAT-iCre rats.

Other related rats

Registered Symbol: LE-Tg(DAT-iCre)1Ottc
RGD ID: 9588572 
Proposed Name:  LE-Tg(DAT::iCre)1Ottc
RRRC#: 758 

Acknowledgements

YaJun Zhang, Julie Necarsulmer, Pyry Koivula, Christopher T. Richie, Brandon Harvey, Janette Lebron