CRISPR/Cas9‐mediated targeting of the <i>Rosa26</i> locus produces Cre reporter rat strains for monitoring Cre<i>–loxP</i>‐mediated lineage tracing

Ma, Yuanwu; Liu, Yu; Pan, Shuo; Gao, Shan; Chen, Wei; Zhang, Xu; Dong, Wei; Li, Jing; Zhou, Rui; Huang, Lan; Han, Yunlin; Bai, Lin; Zhang, Li; Zhang, Lianfeng

doi:10.1111/febs.14188

Cited by 40 publications

(37 citation statements)

References 52 publications

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“…Rather, their technologies have been widely applied to genetic modification studies, including the generation of a ubiquitous mCherry rat. 22 As these exciting research tools were not immediately available due to cost and accessibility for use in our tissue engineering applications, we decided to have the University of Michigan's Transgenic Animal Model Core develop a rat line for our tissue engineering needs. In an effort to increase accessibility to this technology, we will be making our animals available through the Rat Resource & Research Center (RRRC).…”

Section: Introductionmentioning

confidence: 99%

A Transgenic tdTomato Rat for Cell Migration and Tissue Engineering Applications

Syverud

Gumucio

Rodriguez

et al. 2018

Tissue Engineering Part C: Methods

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The growing deficit in suitable tissues for patients awaiting organ transplants demonstrates the clinical need for engineered tissues as alternative graft sources. Demonstrating safety and efficacy by tracking the migration and fate of implanted cells is a key consideration required for approval of promising engineered tissues. Cells from transgenic animals that express green fluorescent protein (GFP) are commonly used for this purpose. However, GFP can create difficulties in practice due to high levels of green autofluorescence in many musculoskeletal tissues. Tandem-dimer tomato (tdTomato) is a stable, robust red fluorescent protein that is nearly threefold brighter than GFP. Our objective was to create a line of transgenic rats that ubiquitously express tdTomato in all cells, driven by the human ubiquitin C promoter. We sought to determine the rats' utility in tissue engineering applications by fabricating engineered skeletal muscle units (SMUs) from isolated muscle-derived tdTomato cells. These tdTomato SMUs were implanted into a volumetric muscle loss (VML) defect of the tibialis anterior muscle in a rat ubiquitously expressing GFP. We also evaluated a novel method for modularly combining individual SMUs to create a larger engineered tissue. Following a recovery period of 28 days, we found that implantation of the modular SMU led to a significant decrease in the size of the remaining VML deficit. Histological analysis of explanted tissues demonstrated both tdTomato and GFP expression in the repair site, indicating involvement of both implanted and host cells in the regeneration process. These results demonstrate the successful generation of a tdTomato transgenic rat, and the use of these rats in tissue engineering and cell migration applications. Furthermore, this study successfully validated a method for scaling engineered tissues to larger sizes, a factor that will be important for repairing volumetric injuries in more clinically relevant models.

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Section: Introductionmentioning

confidence: 99%

A Transgenic tdTomato Rat for Cell Migration and Tissue Engineering Applications

Syverud

Gumucio

Rodriguez

et al. 2018

Tissue Engineering Part C: Methods

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“…The Sprague‐Dawley (SD) Rosa26‐imCherry rats were generated in our lab as reported previously . The floxed eGFP and mCherry cassette was inserted in the Rosa26 locus and the expression of mCherry was induced in the presence of Cre protein, which was used as Cre reporter rat strain.…”

Section: Methodsmentioning

confidence: 99%

Knockout of ISCA1 causes early embryonic death in rats

Yang

Zhang

et al. 2019

Anim Models and Exp Med

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Background Iron‐sulfur cluster assembly 1 ( ISCA 1) is an iron‐sulfur (Fe/S) carrier protein that accepts Fe/S from a scaffold protein and transfers it to target proteins including the mitochondrial Fe/S containing proteins. ISCA 1 is also the newly identified causal gene for multiple mitochondrial dysfunctions syndrome ( MMDS ). However, our knowledge about the physiological function of ISCA 1 in vivo is currently limited. In this study, we generated an ISCA 1 knockout rat line and analyzed the embryo development. Methods ISCA 1 knockout rats were generated by replacing the exon1 of ISCA 1 gene with the mC herry‐Cre fusion gene using CRISPR ‐Cas9 technology. The ISCA 1 expression pattern was analyzed by fluorescence imaging using ISCA 1 promotor driven Cre and mC herry expression. The embryonic morphology was examinated by microscope and mitochondrial proteins were tested by Western blot. Results An ISCA 1 knockout rat line was obtained, which expressed mC herry‐Cre fusion protein. Both of the fluorescence images from mC herry and Cre induced mC herry in a reporter rat strain, showing that ISCA 1 expressed in most of the tissues in rats. The ISCA 1 knockout resulted in abnormal development at 8.5 days, with a significant decrease of NDUFA 9 protein and an increase of aconitase 2 ( ACO 2) in rat embryos. Conclusion Deletion of ISCA 1 induced early death in rats. ISCA 1 affected the expression of key proteins in the mitochondrial respiratory chain complex, suggesting that ISCA 1 has an important influence on the respiratory complex and energy metabolism.

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“…In the near future, the application of new genome editing nucleases, such as meganucleases, 166 ZFNs, 210 TALENs, 211 and CRISPR/Cas9, 212 , 213 will facilitate the development of rats with disease-specific mutations including with Cre-conditional mutations. 214 , 215 …”

Section: Ratsmentioning

confidence: 99%

Humanization of Immunodeficient Animals for the Modeling of Transplantation, Graft Versus Host Disease, and Regenerative Medicine

et al. 2020

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The humanization of animals is a powerful tool for the exploration of human disease pathogenesis in biomedical research, as well as for the development of therapeutic interventions with enhanced translational potential. Humanized models enable us to overcome biologic differences that exist between humans and other species, while giving us a platform to study human processes in vivo. To become humanized, an immune-deficient recipient is engrafted with cells, tissues, or organoids. The mouse is the most well studied of these hosts, with a variety of immunodeficient strains available for various specific uses. More recently, efforts have turned to the humanization of other animal species such as the rat, which offers some technical and immunologic advantages over mice. These advances, together with ongoing developments in the incorporation of human transgenes and additional mutations in humanized mouse models, have expanded our opportunities to replicate aspects of human allotransplantation and to assist in the development of immunotherapies. In this review, the immune and tissue humanization of various species is presented with an emphasis on their potential for use as models for allotransplantation, graft versus host disease, and regenerative medicine.

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CRISPR/Cas9‐mediated targeting of the Rosa26 locus produces Cre reporter rat strains for monitoring Cre–loxP‐mediated lineage tracing

Cited by 40 publications

References 52 publications

A Transgenic tdTomato Rat for Cell Migration and Tissue Engineering Applications

A Transgenic tdTomato Rat for Cell Migration and Tissue Engineering Applications

Knockout of ISCA1 causes early embryonic death in rats

Humanization of Immunodeficient Animals for the Modeling of Transplantation, Graft Versus Host Disease, and Regenerative Medicine

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