scholarly journals A CRISPR/Cas9 genome editing pipeline in the EndoC-βH1 cell line to study genes implicated in beta cell function

2020 ◽  
Vol 4 ◽  
pp. 150 ◽  
Author(s):  
Antje K. Grotz ◽  
Fernando Abaitua ◽  
Elena Navarro-Guerrero ◽  
Benoit Hastoy ◽  
Daniel Ebner ◽  
...  

Type 2 diabetes (T2D) is a global pandemic with a strong genetic component, but most causal genes influencing the disease risk remain unknown. It is clear, however, that the pancreatic beta cell is central to T2D pathogenesis. In vitro gene-knockout (KO) models to study T2D risk genes have so far focused on rodent beta cells. However, there are important structural and functional differences between rodent and human beta cell lines. With that in mind, we have developed a robust pipeline to create a stable CRISPR/Cas9 KO in an authentic human beta cell line (EndoC-βH1). The KO pipeline consists of a dual lentiviral sgRNA strategy and we targeted three genes (INS, IDE, PAM) as a proof of concept. We achieved a significant reduction in mRNA levels and complete protein depletion of all target genes. Using this dual sgRNA strategy, up to 94 kb DNA were cut out of the target genes and the editing efficiency of each sgRNA exceeded >87.5%. Sequencing of off-targets showed no unspecific editing. Most importantly, the pipeline did not affect the glucose-responsive insulin secretion of the cells. Interestingly, comparison of KO cell lines for NEUROD1 and SLC30A8 with siRNA-mediated knockdown (KD) approaches demonstrate phenotypic differences. NEUROD1-KO cells were not viable and displayed elevated markers for ER stress and apoptosis. NEUROD1-KD, however, only had a modest elevation, by 34%, in the pro-apoptotic transcription factor CHOP and a gene expression profile indicative of chronic ER stress without evidence of elevated cell death. On the other hand, SLC30A8-KO cells demonstrated no reduction in KATP channel gene expression in contrast to siRNA silencing. Overall, this strategy to efficiently create stable KO in the human beta cell line EndoC-βH1 will allow for a better understanding of genes involved in beta cell dysfunction, their underlying functional mechanisms and T2D pathogenesis.

2019 ◽  
Vol 4 ◽  
pp. 150 ◽  
Author(s):  
Antje K. Grotz ◽  
Fernando Abaitua ◽  
Elena Navarro-Guerrero ◽  
Benoit Hastoy ◽  
Daniel Ebner ◽  
...  

Type 2 diabetes (T2D) is a global pandemic with a strong genetic component, but most causal genes influencing the disease risk remain unknown. It is clear, however, that the pancreatic beta cell is central to T2D pathogenesis. In vitro gene-knockout (KO) models to study T2D risk genes have so far focused on rodent beta cells. However, there are important structural and functional differences between rodent and human beta cell lines. With that in mind, we have developed a robust pipeline to create a stable CRISPR/Cas9 KO in an authentic human beta cell line (EndoC-βH1). The KO pipeline consists of a dual lentiviral sgRNA strategy and we targeted three genes (INS, IDE, PAM) as a proof of concept. We achieved a significant reduction in mRNA levels and complete protein depletion of all target genes. Using this dual sgRNA strategy, up to 94 kb DNA were cut out of the target genes and the editing efficiency of each sgRNA exceeded >87.5%. Sequencing of off-targets showed no unspecific editing. Most importantly, the pipeline did not affect the glucose-responsive insulin secretion of the cells. Interestingly, comparison of KO cell lines for NEUROD1 and SLC30A8 with siRNA-mediated knockdown (KD) approaches demonstrate phenotypic differences. NEUROD1-KO cells were not viable and displayed elevated markers for ER stress and apoptosis. NEUROD1-KD, however, only had a modest elevation, by 34%, in the pro-apoptotic transcription factor CHOP and a gene expression profile indicative of chronic ER stress without evidence of elevated cell death. On the other hand, SLC30A8-KO cells demonstrated no reduction in KATP channel gene expression in contrast to siRNA silencing. Overall, this strategy to efficiently create stable KO in the human beta cell line EndoC-βH1 will allow for a better understanding of genes involved in beta cell dysfunction, their underlying functional mechanisms and T2D pathogenesis.


2009 ◽  
Vol 53 (12) ◽  
pp. 5127-5133 ◽  
Author(s):  
Jonas Steinmann ◽  
Skarphéđinn Halldórsson ◽  
Birgitta Agerberth ◽  
Gudmundur H. Gudmundsson

ABSTRACT Antimicrobial peptides (AMPs) are important components of our first line of defense. Induction of AMPs such as LL-37 of the cathelicidin family might provide a novel approach in treating bacterial infections. In this study we identified 4-phenylbutyrate (PBA) as a novel inducer of AMP expression and investigated affected regulatory pathways. We treated various cell lines with PBA and assessed mRNA expression by real-time reverse transcriptase PCR (RT-PCR). Cathelicidin AMP (CAMP) gene expression was found to be upregulated in all four cell lines tested. Additionally, we found that the beta-defensin 1 gene was upregulated in the lung epithelial cell line VA10 while being downregulated in the monocytic cell line U937. Further we found that PBA induced CAMP gene expression synergistically with 1,25-dihydroxyvitamin D3 at both protein and mRNA levels. The general mechanism of induction of CAMP gene expression by PBA was found to be dependent on protein synthesis. Results from quantitative chromatin immunoprecipitation experiments challenge the common view that histone deacetylase inhibitors directly increase CAMP gene expression. Furthermore, we have demonstrated that inhibition of the mitogen-activated protein kinases MEK1/2 and c-Jun N-terminal kinase attenuate PBA-induced CAMP gene expression. Similarly, α-methylhydrocinnamate (ST7), an analogue of PBA, increases CAMP gene expression. Our findings contribute to understanding of the regulation of AMP expression and suggest that PBA and/or ST7 is a promising drug candidate for treatment of microbial infections by strengthening the epithelial antimicrobial barriers.


Diabetologia ◽  
1995 ◽  
Vol 38 (8) ◽  
pp. 927-935
Author(s):  
M. Asfari ◽  
W. De ◽  
M. N�el ◽  
P. E. Holthuizen ◽  
P. Czernichow

2021 ◽  
Vol 224 (2) ◽  
pp. S557-S558
Author(s):  
Callie Reeder ◽  
Kendra Sylvester ◽  
Walker Hagan ◽  
Fatima Zahra Aly ◽  
Maurine Keller-wood ◽  
...  

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