LE-Tg(DIO-iRFP)3Ottc

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

Background

Genetically-encoded fluorescent reporters are used in the biological sciences for the identification and observation of cells that express transgenic proteins. Near-infrared (NIR) fluorescent proteins have superior light penetration through biological tissue, but are not yet widely adopted. Using the near-infrared fluorescent protein, iRFP713, improves the imaging resolution in thick tissue sections or the intact brain due to the reduced light-scattering at the longer, NIR wavelengths used to image the protein. Additionally, iRFP713 can be used to identify transgenic cells without photobleaching other fluorescent reporters or affecting opsin function for optogenetic experiments. We have generated a transgenic rat (Long Evans background) that expresses iRFP713 in a Cre-dependent manner.

Status and Availability

This rat has been published (PMID: 28380331).

As of May 30, 2017, this strain is available as line #747 at RRRC. 

This rat is registered at the Rat Genome Database (RGD) as RGD ID#9588559. 

Transgene Information

Figure 1.  Schematic of the DIO-iRFP transgene.  The insert encoding iRFP713 was amplified with linkered oligos and Addgene #31857 as a template. This insert was recombined into the backbone (pAAV EF1a DIO EYFP, Addgene 27056, digested with NheI and AscI restriction enzymes) using In-Fusion cloning mix (Clontech) to produce pOTTC374 (Addgene #47626).  The pOTTC374 (pAAV EF1a DIO iRFP) plasmid was digested with MluI and RsrII, gel purified away from the plasmid backbone, and microinjected into fertilized oocytes harvested from a Long Evans rat by the NIMH Transgenic Core.  Surviving pups were screened for the integrated transgene by PCR genotyping.  This line (LE-Tg(DIO-iRFP)3Ottc) has 6 copies of the transgene per copy of Ggt1 (# copy per haploid genome) as determined by droplet digital PCR.

Phenotypic Characterization

Figure 2. DIO-iRFP transgenic rats express iRFP713 in a Cre-dependent manner in three different brain regions. Representative images of the iRFP expression in phenotypically positive Long-Evans DIO-iRFP transgenic rats. DIO-iRFP line 3(Long-Evans) rats were injected in the prefrontal cortex (left), striatum (middle), and midbrain (right) with AAV1-GFP-Cre. Immunohistochemistry for NeuN was used to indicate neurons, which colocalized with iRFP expression. Scale bars are 50 μm. Download print resolution version here.

Figure 2 was modified and reprinted from “Richie et al. Near-infrared fluorescent protein iRFP713 as a reporter protein for optogenetic vectors, a transgenic Cre-reporter rat, and other neuronal studies. J Neurosci Methods. 2017 Jun 1;284:1-14.” With permission from Elsevier.

For more details and data on phenotypic characterization, see reference (PMID: 28380331).

Breeding Strategy

Breeding Information, click here for PDF

Genotyping Assays

Assay for presence of EF1a and iRFP, click here for PDF

References that cite this rat

@article{Richie:2017aa,

title = {Near-infrared fluorescent protein iRFP713 as a reporter protein for optogenetic vectors, a transgenic Cre-reporter rat, and other neuronal studies.},

author = {Christopher T Richie and Leslie R Whitaker and Keith W Whitaker and Julie Necarsulmer and Heather A Baldwin and Yajun Zhang and Lowella Fortuno and Josh J Hinkle and Pyry Koivula and Mark J Henderson and Wenzhi Sun and Kai Wang and Jeffrey C Smith and Jim Pickel and Na Ji and Bruce T Hope and Brandon K Harvey},

url = {https://www.ncbi.nlm.nih.gov/pubmed/28380331},

doi = {10.1016/j.jneumeth.2017.03.020},

issn = {1872-678X (Electronic); 0165-0270 (Linking)},

year  = {2017},

date = {2017-04-02},

urldate = {2017-04-02},

journal = {J Neurosci Methods},

volume = {284},

pages = {1--14},

address = {Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD 21224, United States.},

abstract = {BACKGROUND: The use of genetically-encoded fluorescent reporters is essential for the identification and observation of cells that express transgenic modulatory proteins. Near-infrared (NIR) fluorescent proteins have superior light penetration through biological tissue, but are not yet widely adopted. NEW METHOD: Using the near-infrared fluorescent protein, iRFP713, improves the imaging resolution in thick tissue sections or the intact brain due to the reduced light-scattering at the longer, NIR wavelengths used to image the protein. Additionally, iRFP713 can be used to identify transgenic cells without photobleaching other fluorescent reporters or affecting opsin function. We have generated a set of adeno-associated vectors in which iRFP713 has been fused to optogenetic channels, and can be expressed constitutively or Cre-dependently. RESULTS: iRFP713 is detectable when expressed in neurons both in vitro and in vivo without exogenously supplied chromophore biliverdin. Neuronally-expressed iRFP713 has similar properties to GFP-like fluorescent proteins, including the ability to be translationally fused to channelrhodopsin or halorhodopsin, however, it shows superior photostability compared to EYFP. Furthermore, electrophysiological recordings from iRFP713-labeled cells compared to cells labeled with mCherry suggest that iRFP713 cells are healthier and therefore more stable and reliable in an ex vivo preparation. Lastly, we have generated a transgenic rat that expresses iRFP713 in a Cre-dependent manner. CONCLUSIONS: Overall, we have demonstrated that iRFP713 can be used as a reporter in neurons without the use of exogenous biliverdin, with minimal impact on viability and function thereby making it feasible to extend the capabilities for imaging genetically-tagged neurons in slices and in vivo.},

note = {*First paper describing LE-Tg (DIO-iRFP)3Ottc rat.

*First paper describing LE-Tg (DIO-iRFP)9Ottc rat.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

Blog/Comments/Reviews

No comments or reviews are available at this time.

Other related rats

Registered Symbol: SD-Tg(DIO-iRFP)9Ottc
RGD ID: 11081153 
Proposed Name: SD-Tg(DIO-iRFP)9Ottc
RRRC#: 748 

*Rat Research and Resource Center (RRRC)

Acknowledgements

YaJun Zhang, Julie Necarsulmer, Chris Richie, Brandon Harvey, Janette Lebron