scholarly journals Cell type–specific manipulation with GFP-dependent Cre recombinase

2015 ◽  
Vol 18 (9) ◽  
pp. 1334-1341 ◽  
Author(s):  
Jonathan C Y Tang ◽  
Stephanie Rudolph ◽  
Onkar S Dhande ◽  
Victoria E Abraira ◽  
Seungwon Choi ◽  
...  
Keyword(s):  
Cre Recombinase ◽  
Cell Type ◽  
Cell Type Specific

scholarly journals Zebrafish Cre/lox regulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation

2021 ◽  
Author(s):  
Maira P. Almeida ◽  
Sekhar Kambakam ◽  
Fang Liu ◽  
Zhitao Ming ◽  
Jordan M. Welker ◽  
...  
Keyword(s):  
Gene Function ◽  
Cre Recombinase ◽  
Gene Trap ◽  
Gene Inactivation ◽  
Loss Of Function ◽  
Cell Type ◽  
Active Gene ◽  
Indel Mutation ◽  
Targeted Integration ◽  
Cell Type Specific

The ability to regulate gene activity spatially and temporally is essential to investigate cell type specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish, but simple and efficient methods for isolation of stable, Cre/lox regulated alleles are lacking. Here we applied our GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to generate floxed conditional alleles that provide robust gene knockdown and strong loss of function phenotypes. A universal targeting vector, UFlip, with sites for cloning short 24-48 bp homology arms flanking a floxed mRFP gene trap plus secondary reporter cassette, was integrated into an intron in hdac1, rbbp4, and rb1. Active, gene off orientation hdac1-UFlip-Off and rb1-UFlip-Off integration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientation rbbp4-UFlip-On and rb1-UFlip-On integration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs of loxP and lox2272 sites, inverting and locking the cassette into the active, gene off orientation, and the expected mutant phenotypes. In combination with our endogenous neural progenitor Cre drivers we demonstrate rbbp4-UFlip-On and rb1-UFlip-On gene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP in rbbp4-UFlip-Off, or 2A-KalTA4 in rb1-UFlip-Off, shows strong RFP expression in wild type or UAS:RFP injected embryos, respectively. Together these results validate a simplified approach for efficient isolation of highly mutagenic Cre/lox responsive conditional gene alleles to advance zebrafish Cre recombinase genetics.

scholarly journals Characterization of Cre recombinase  mouse lines enabling cell type‐specific targeting of postnatal intervertebral discs

2019 ◽  
Vol 234 (9) ◽  
pp. 14422-14431 ◽  
Author(s):  
Yixin Zheng ◽  
Xuejie Fu ◽  
Qingbai Liu ◽  
Shengqi Guan ◽  
Cunchang Liu ◽  
...  
Keyword(s):  
Cre Recombinase ◽  
Intervertebral Discs ◽  
Cell Type ◽  
Specific Targeting ◽  
Cell Type Specific ◽  
Mouse Lines

scholarly journals Inflammation and Tissue Damage Mediated By PAR-1 Expression Is Cell Type-Specific and Varies Depending on the Inflammatory Challenge

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 133-133
Author(s):  
Alexander A Boucher ◽  
Leah A. Rosenfeldt ◽  
Duaa Mureb ◽  
Anna K Kopec ◽  
James P. Luyendyk ◽  
...  
Keyword(s):  
Weight Loss ◽  
Liver Injury ◽  
Cre Recombinase ◽  
Colonic Crypt ◽  
Cell Type ◽  
Infectious Colitis ◽  
Effector Cells ◽  
Cell Type Specific ◽  
Hepatocyte Necrosis

Abstract Protease activated receptor-1 (PAR-1) is a G protein-coupled receptor activated by cleavage of the extracellular domain, revealing an encrypted ligand. PAR-1 can be activated by several proteases, including thrombin and activated protein C, coupling hemostatic proteases to cell signaling. PAR-1 activation is a significant determinant of the inflammatory response in multiple settings. However, the fact that PAR-1 is expressed on many cell types makes a mechanistic understanding of the effector mechanisms challenging. We hypothesized that the effector cells expressing PAR-1 that drive inflammatory functions are contextually dependent on the type of inflammatory challenge. To test this hypothesis, we used recently-generated mice carrying a conditional (floxed) PAR-1 allele (PAR-1fl/fl) interbred with established transgenic mice expressing Cre recombinase in a cell type-specific manner in two experimental mouse models, an acetaminophen (APAP) hepatotoxicity model and a Citrobacter rodentium infectious colitis model. Cre recombinase was expressed under the control of the CMV promoter (global PAR-1 deletion), the Tie2 promoter (endothelial and hematopoietic cell PAR-1 deletion), and the LysM promoter (myeloid PAR-1 deletion). An established hepatocyte-tropic AAV Cre recombinase vector was used to delete hepatocyte-associated PAR-1. To define the cell type-specific role of PAR-1 in APAP induced liver damage, mice were challenged with APAP and euthanized 24 hours later for analyses. Control mice exposed to toxic APAP doses had a 10-100 fold increase in serum alanine transaminase (ALT) levels and ~50% hepatocyte necrosis based on morphometric analyses of liver tissue sections. Consistent with previous results, APAP-challenged mice with global PAR-1 deletion (CMV Cre/PAR-1fl/fl) demonstrated significantly lower serum ALT levels relative to control animals (PAR-1fl/fl) and significantly less hepatocyte necrosis. Neither AAV-Cre mediated deletion of PAR-1 in hepatocytes nor LysM Cre-mediated deletion in myeloid cells had any significant impact on APAP-induced liver injury. In contrast, deletion of PAR-1 in the endothelial and hematopoietic compartments (Tie2 Cre) phenocopied global PAR-1 deletion, resulting in a significant diminution in serum ALT and a 30% decrease in APAP-induced hepatocyte necrosis. Together, these results suggest that either a non-myeloid leukocyte and/or endothelial cells are the effector cells expressing PAR-1 that promote liver damage in this experimental context. To define the role of PAR-1 in the setting of infectious colitis, mice were challenged with C. rodentium by oral gavage and euthanized 14 days later for analyses. Global deletion of PAR-1 resulted in significantly less weight loss, longer colonic crypts, and a local diminution in multiple inflammatory cytokines (IFNγ, IL-1β, IL-17A, IL-22, and IL-6). In contrast to what was observed in APAP-induced liver injury, cell type-specific deletion of PAR-1 in myeloid cells (LysM Cre) resulted in a phenocopy of global PAR-1 deficiency, including blunted weight loss and longer colonic crypt lengths. The difference in colonic crypt length appeared to be due, at least in part, to genotype-dependent differences in colonic epithelial apoptosis, demonstrated by diminished staining for cleaved caspase 3 in crypts harvested from LysM Cre/PAR-1fl/fl mice relative to controls. Deletion of myeloid-associated PAR-1 also resulted in a local diminution in several cytokines (IFNγ, IL-1β, IL-17A, and IL-6) similar to global PAR-1 deficiency. Together, these results strengthen previous data showing the importance of PAR-1 in mediating multiple inflammatory pathologies. These data also highlight the finding that the specific effector cells expressing PAR-1 that promote inflammatory processes may differ depending on the type of inflammatory challenge. A detailed effector cell-specific understanding of the role of PAR-1 is critical to understanding the mechanisms coupling hemostatic proteases to inflammation and tissue injury and for defining the potential of PAR-1 as a target to mitigate these pathologies. Disclosures Palumbo: Ionis Pharmaceuticals: Research Funding.

IFIH1-deficiency results in a cell-type specific alteration of autophagic activity in response to S. typhimurium

2017 ◽  
Vol 55 (05) ◽  
pp. e28-e56
Author(s):  
S Macheiner ◽  
R Gerner ◽  
A Pfister ◽  
A Moschen ◽  
H Tilg
Keyword(s):  
Cell Type ◽  
A Cell ◽  
Cell Type Specific ◽  
Autophagic Activity ◽  
Specific Alteration
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