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.
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.
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).
References that cite this rat:
*Richie CT, Whitaker LR, Whitaker KW, Necarsulmer J, Baldwin HA, Zhang Y, Fortuno L, Hinkle JJ, Koivula P, Henderson MJ, Sun W, Wang K, Smith JC, Pickel J, Ji N, Hope BT, Harvey BK. 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. doi: 10.1016/j.jneumeth.2017.03.020. Epub 2017 Apr 2. (PMID: 28380331).
*First paper describing LE-Tg (DIO-iRFP)3Ottc rat.
No comments or reviews are available at this time.
Other related rats:
*Rat Research and Resource Center (RRRC)
Acknowledgements: YaJun Zhang, Julie Necarsulmer, Chris Richie, Brandon Harvey, Janette Lebron