198 PROGESTERONE, ESTROGEN (ER-α AND ER-β), AND OXYTOCIN RECEPTOR GENE EXPRESSION IN CANINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 248
Author(s):  
A. A. P. Derussi ◽  
A. C. S. Castilho ◽  
R. W. A. Souza ◽  
R. Volpato ◽  
C. R. F. Guaitolini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Hormone Receptors ◽  
Target Genes ◽  
Receptor Gene ◽  
Rna Extraction ◽  
Amplification Efficiency ◽  
Mrna Levels ◽  
Lh Surge ◽  
Total Rna

The aim of this study was to compare the mRNA levels of hormone receptor for progesterone (PR), oestrogen α (ER-α), oestrogen β (ER-β), and oxytocin (OTR) in canine morulae and blastocysts. Ten healthy mature bitches were inseminated based on monitoring vaginal cytology and progesterone concentration. The first insemination was performed on Day 2 after the preovulatory LH surge (progesterone 4 ng mL–1), and the second was performed 48 h later. All females were submitted to ovariohysterectomy (OVH), and the oviduct as well the uterurs were flushed with PBS solution to obtain the embryos. The females were divided into two groups: Group A (n = 5), morulae were collected 8 days after the LH surge and Group B (n = 5), blastocysts were collected 12 days after the LH surge. The pools (n = 10) of embryos (5 embryos/pool) were stored in RNAlater® (Ambion, Life Technologies, USA) at –80°C. The samples were analysed together. The RNA later was removed used PBS calcium free and the total RNA extraction was performed using the Qiagen RNeasy micro-kit (Hildesheim, Germany). Before reverse-transcription (RT) reaction, the total RNA was treated with DNase I Amplification Grade (Invitrogen Life Technologies, Carlsbad, CA, USA). The gene expression of target genes was assessed by real-time RT-qPCR, using SuperScript III for RT and power SYBR Green PCR Master Mix (Applied Biosystems, USA) for cDNA for PCR. The primers for target genes were designed using the software Primer Express® (Applied Biosystems, USA). The gene expression of target genes was normalized by HPRT gene and the relative abundance of mRNA was determined by the ΔΔct method corrected by amplification efficiency using Pffafl’s equation. The means of mRNA relative abundance were compared by t-test. The PR mRNA expression only in blastocysts is similar to the results obtained by Hou et al. (1997) in rat embryos. It is believed that the absence of PR in the early stages of cleavage is due to the indirect action of progesterone by growth factors produced by the maternal reproductive tract (2). Apparently, ER-β action does not occur in the embryo canine phases analysed; however, the action of ER-α seems related to the deployment signal as seen by Hou et al. (1996) in rats. Similarly to findings in the literature, OTR expression decreased in canine embryonic development. This receptor was produced by blastocysts while present in the uterus, which may represent an incidental mechanism to the embryo control of endometrial receptivity, such as also to prevent the development of endometrial luteolytic mechanism. The variation in hormone receptors gene expression in canine embryos can be influencing the transition from morula to blastocyst. In addition, a hormonal influence on these structures can occur in different ways.

scholarly journals Evaluation of Different RNA Extraction Methods from Agropatch Suppressor Assay for Small Quantities of Plant Tissue and Their Application for Analysis of Gene Expression

2018 ◽  
Vol 10 (3) ◽  
pp. 348-353
Author(s):  
Amir G. SHAHRIARI ◽  
Aminallah TAHMASEBI
Keyword(s):  
Gene Expression ◽  
Plant Tissue ◽  
Target Genes ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Total Rna ◽  
Expression Studies ◽  
Rna Extraction Methods ◽  
Gene Expression Studies ◽  
Commercial Kit

The agroinfiltration assay provides fast and efficient way to transiently express genes into plant cells by Agrobacterium tumefaciens. Extraction of RNA of high quality and sufficient amounts is prerequisite for gene expression studies such as quantitative Real Time PCR (q-PCR) from infiltrated areas in agropatch suppressor assay with small quantities of plant tissue. To attain prime RNA extraction from small tissues of infiltrated N. benthamiana plants with Potato virus A helper component proteinase viral suppressor protein, the efficiency of three RNA extraction methods (LiCl, TRIzol reagent and commercial kit) was evaluated. The total RNA yield with LiCl method was 2.83 and 33.2-fold greater than that of TRIzol reagent and commercial kit, respectively. Also, total RNA yield using TRIzol reagent was 11.7-fold higher than that with commercial kit. The A260/A280 ratio mean for TRI reagent (1.95) and kit (1.9) extractions were within the optimum range.q-PCR revealed that the cycle threshold values of housekeeping gene, EIF-1α and target genes AGO1 and ATG6 for RNA extracted using LiCl and kit were 1.07 to 1.3 and 1.02 to 1.12 times higher than those evaluated with the TRIzol method. Overall, TRIzol method showed the most effective approach for obtaining RNA from N. benthamiana patches in gene expression studies.

180 HEAT STRESS INDUCES ALTERATION IN EXPRESSION OF GENES RELATED TO COMPETENCE AND IMPLANTATION IN NELORE BOVINE IN VITRO-PRODUCED EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 191
Author(s):  
C. F. Silva ◽  
A. C. S. Castilho ◽  
R. A. Satrapa ◽  
R. Z. Puelker ◽  
E. M. Razza ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Internal Control ◽  
Target Genes ◽  
Rna Extraction ◽  
Bos Indicus ◽  
Gene Expression Pattern ◽  
Mrna Levels ◽  
Stressed Group

Several factors affect early embryonic development in cattle, including heat stress. These factors can contribute to high early embryonic loss, probably altering gene expression. Studies using microarray-profiled genome-wide RNA expression for in vitro-produced blastocysts have compared embryos resulting in calf delivery or no pregnancy, and they have identified genes with potential roles in pregnancy and embryo competence. The aim of the present work was to compare the expression of some genes (PLAC8, HSF1, COX-2, and CDX-2) related to embryo competence and embryonic implantation between in vitro-produced embryos from the Nelore breed (Bos indicus), submitted or not submitted to heat stress. Oocytes from Nelore cows were aspirated by ovum pickup and matured for 22 h (TCM-199 with bicarbonate, supplemented with 10% FCS, 2 μL mL–1 of pyruvate, 75 μg mL–1 of amicacin, 20 μg mL–1 of FSH, and 2 IU mL–1 of hCG) at 38.5°C with 5% CO2 in air. The fertilization (Day 0) was performed with semen from Nellore bulls. After a 12-h fertilization period, in Tyrode’s lactate stock medium supplemented with 6 mg mL–1 of BSA, 2 mL mL–1 of pyruvate, 75 mg mL–1 of amicacin, 11 mg mL–1 of heparin, and 44 mL mL–1 of phenylalanine solution, presumptive zygotes were denuded and randomly divided in 2 groups: nonstressed and stressed. The culture medium was SOFaaci supplemented with sodium pyruvate (0, 2%), 5 mg mL–1 of BSA and 5% FCS. Embryo culture was performed at 38.5°C, 90% N2, 5% CO2, and 5% O2. In the stressed group, 96 h after fertilization, the embryos were subjected to heat stress of 41°C for 6 consecutive hours and then returned to a temperature of 38.5°C. On Day 7, pools of 5 blastocysts (nonstressed, n = 9; stressed, n = 7) were submitted to total RNA extraction (RNeasy, Qiagen, Valencia, CA). The gene expression of target genes was measured by real-time RT-PCR with oligo-dT in the reverse transcription and bovine-specific primers in the PCR. Expression of cyclophlin A was used as an internal control. The means of mRNA levels of target genes between the groups were compared by t-test. The PLAC8 mRNA levels were higher in nonstressed blastocysts in comparison with the stressed group. The HSF1 and CDX2 mRNA was detectable only in nonstressed embryos. The COX2 mRNA levels did not differ between groups. The higher levels of PLAC8 and the CDX2 expression on nonstressed embryos indicate better competence of embryos not submitted to heat stress. Furthermore, the absence of HSF1 mRNA in the stressed embryos does not reflect the lack of biological activity of this protein. In conclusion, the data indicate that heat stress alters the gene expression pattern of in vitro-produced embryos in the Nelore breed. FAPESP (São Paulo, Brazil) is acknowledged for funding and fellowships for Castilho, Satrapa, and Razza.

137 DIFFERENTIAL GENE EXPRESSION OF IN VITRO-MATURED BOVINE OOCYTES WITH OR WITHOUT A POLAR BODY

2012 ◽  
Vol 24 (1) ◽  
pp. 181
Author(s):  
M. M. Pereira ◽  
S. Wohlres-Viana ◽  
J. N. S. Sales ◽  
A. R. Camargo ◽  
C. C. R. Quintão ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Relative Abundance ◽  
Polar Body ◽  
Rna Extraction ◽  
Mrna Levels ◽  
Insulin Like Growth Factor ◽  
Glucose Transporter 1 ◽  
Bovine Oocytes

Oocyte competence is associated with the amount of transcripts stored in the ooplasm and oocyte ability to extrude polar bodies (PB). To our knowledge, however, no data comparing mRNA levels between bovine oocytes maturated in vitro with or without PB are available. The aim of the present study was to compare the relative abundance of transcripts of glucose transporter 1 (GLUT1), insulin-like growth factor 1 receptor (IGF1R), insulin-like growth factor 2 receptor (IGF2R), growth differentiation factor-9 (GDF9) and aquaporin 3 (AQP3) genes between oocytes with and without PB (PB and NPB groups, respectively) following in vitro maturation. Immature bovine oocytes were obtained by follicular aspiration and matured in TCM-199 (Gibco Life Technologies, New York, NY, USA) containing 10% of oestrus cow serum and 20 μg mL–1 of FSH (Pluset, Serono, Italy) for 24 h under 5% CO2 in air at 38.5°C. Subsequently, oocytes were visually classified according to the presence or absence of PB and then denuded and rapidly frozen in liquid nitrogen. Three pools of 10 oocytes for each group were subjected to total RNA extraction using the RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions and treated with DNase. Reverse transcription and cDNA amplification were performed using the TransPlex Complete Whole Transcriptome Amplification Kit (WTA2, Sigma, St. Louis, MO, USA) according to the manufacturer's instructions. Relative abundance of the target transcripts was performed by quantitative RT-PCR (Applied Biosystems Prism 7300 Sequence Detection Systems, Foster City, CA, USA) using a mixture of SYBR® Green PCR Master Mix (Applied Biosystems), 200 ng of cDNA, nuclease-free water and specific primers for each reaction. Expression of the β-actin gene was used as an endogenous reference. Relative gene expression analysis was performed using the software REST© 2005 using the Pair Wise Fixed Reallocation Randomization Test©. The relative expression values are presented as mean ± standard error. The relative abundance of GLUT1 (0.81 ± 0.07), IGF2R (0.72 ± 0.07) and GDF9 (0.82 ± 0.10) genes was lower (P < 0.05) for NPB oocytes. There was no difference (P > 0.05) in relative abundance between PB and NPB groups for the other genes. The results suggest that the amount of some transcripts stored in the matured ooplasm is associated with the presence of PB. The authors acknowledge FAPEMIG, CNPq and Innovation Network Project on Animal Reproduction (01.07.01.002).

scholarly journals Identification of RISC-Associated Adenoviral MicroRNAs, a Subset of Their Direct Targets, and Global Changes in the Targetome upon Lytic Adenovirus 5 Infection

2014 ◽  
Vol 89 (3) ◽  
pp. 1608-1627 ◽  
Author(s):  
Florian Bellutti ◽  
Maximilian Kauer ◽  
Doris Kneidinger ◽  
Thomas Lion ◽  
Reinhard Klein
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
A549 Cells ◽  
Global Changes ◽  
Cellular Mirnas ◽  
Total Rna ◽  
Cellular Mrnas ◽  
Comprehensive Picture ◽  
Infected Cells ◽  
Adenovirus 5

ABSTRACTAdenoviruses encode a set of highly abundant microRNAs (mivaRNAs), which are generated by Dicer-mediated cleavage of the larger noncoding virus-associated RNAs (VA RNAs) I and II. We performed deep RNA sequencing to thoroughly investigate the relative abundance of individual single strands of mivaRNA isoforms in human A549 cells lytically infected with human adenovirus 5 (Ad5) at physiologically relevant multiplicities of infection (MOIs). In addition, we investigated their relative abundance in the endogenous RNA-induced silencing complexes (RISCs). The occupation of endogenous RISCs by mivaRNAs turned out to be pronounced but not as dominant as previously inferred from experiments with AGO2-overexpressing cells infected at high MOIs. In parallel, levels of RISC-incorporated mRNAs were investigated as well. Analysis of mRNAs enriched in RISCs in Ad5-infected cells revealed that only mRNAs with complementarity to the seed sequences of mivaRNAs derived from VA RNAI but not VA RNAII were overrepresented among them, indicating that only mivaRNAs derived from VA RNAI are likely to contribute substantially to the posttranscriptional downregulation of host gene expression. Furthermore, to generate a comprehensive picture of the entire transcriptome/targetome in lytically infected cells, we determined changes in cellular miRNA levels in both total RNA and RISC RNA as well, and bioinformatical analysis of mRNAs of total RNA/RISC fractions revealed a general, genome-wide trend toward detargeting of cellular mRNAs upon infection. Lastly, we identified the direct targets of both single strands of a VA RNAI-derived mivaRNA that constituted one of the two most abundant isoforms in RISCs of lytically infected A549 cells.IMPORTANCEViral and cellular miRNAs have been recognized as important players in virus-host interactions. This work provides the currently most comprehensive picture of the entire mRNA/miRNA transcriptome and of the complete RISC targetome during lytic adenovirus infection and thus represents the basis for a deeper understanding of the interplay between the virus and the cellular RNA interference machinery. Our data suggest that, at least in the model system that was employed, lytic infection by Ad5 is accompanied by a measurable global net detargeting effect on cellular mRNAs, and analysis of RISC-associated viral small RNAs revealed that the VA RNAs are the only source of virus-encoded miRNAs. Moreover, this work allows to assess the power of individual viral miRNAs to regulate cellular gene expression and provides a list of proven and putative direct targets of these miRNAs, which is of importance, given the fact that information about validated targets of adenovirus-encoded miRNAs is scarce.

scholarly journals Characterization and Regulation of Type A Endothelin Receptor Gene Expression in Bovine Luteal Cell Types

Endocrinology ◽  
1999 ◽  
Vol 140 (5) ◽  
pp. 2110-2116 ◽  
Author(s):  
Roni Mamluk ◽  
Nitzan Levy ◽  
Bo Rueda ◽  
John S. Davis ◽  
Rina Meidan
Keyword(s):  
Gene Expression ◽  
Receptor Gene ◽  
Cell Types ◽  
Cell Populations ◽  
Mrna Levels ◽  
Factor I ◽  
Receptor Mrna ◽  
Progesterone Production ◽  
Eta Receptor ◽  
Receptor Gene Expression

Abstract Our previous studies demonstrated that endothelin-1 (ET-1), a 21-amino acid vasoconstrictor peptide, has a paracrine regulatory role in bovine corpus luteum (CL). The peptide is produced within the gland where it inhibits progesterone production by acting via the selective type A endothelin (ETA) receptors. The present study was designed to characterize ETA receptor gene expression in different ovarian cell types and its hormonal regulation. ETA receptor messenger RNA (mRNA) levels were high in follicular cells as well as in CL during luteal regression. At this latter stage, high ETA receptor expression concurred with low prostaglandin F2α receptor mRNA. The ETA receptor gene was expressed by all three major cell populations of the bovine CL; i.e. small and large luteal cells, as well as in luteal endothelial cells. Among these various cell populations, the highest ETA receptor mRNA levels were found in endothelial cells. cAMP elevating agents, forskolin and LH, suppressed ETA receptor mRNA expression in luteinized theca cells (LTC). This inhibition was dose dependent and was evident already after 24 h of incubation. In luteinized granulosa cells (LGC), 10 and 100 ng/ml of insulin-like growth factor I and insulin (only at a concentration of 2000 ng/ml) markedly decreased ETA receptor mRNA levels. In both LGC and LTC there was an inverse relationship between ETA receptor gene expression and progesterone production; insulin (in LGC) and forskolin (in LTC) enhanced progesterone production while inhibiting ETA receptor mRNA levels. Our findings may therefore suggest that, during early stages of luteinization when peak levels of both LH and insulin-like growth factor I exist, the expression of ETA receptors in the gland are suppressed. This study demonstrates physiologically relevant regulatory mechanisms controlling ETA receptor gene expression and further supports the inhibitory role of ET-1 in CL function.

scholarly journals NPY and sbGnRH gene expression in juvenile and adult male Brazilian flounder Paralichthys orbignyanus

2011 ◽  
Vol 41 (11) ◽  
pp. 1927-1930 ◽  
Author(s):  
Vinicius Farias Campos ◽  
Tiago Veiras Collares ◽  
Fabiana Kömmling Seixas ◽  
João Carlos Deschamps ◽  
Luis Fernando Fernandes Marins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Extraction ◽  
Sea Bream ◽  
Mrna Levels ◽  
Adult Males ◽  
Adult Fish ◽  
Cdna Synthesis ◽  
Hypothalamic Regulation ◽  
Measure Gene Expression ◽  
Paralichthys Orbignyanus

The objective of this study was to evaluate neuropeptide Y (NPY) and sea bream gonadotropin-release hormone (sbGnRH) gene expression in juvenile and adult males of Brazilian flounder. Hypothalamuses from fish were sampled for total RNA extraction. After cDNA synthesis, real-time PCR was used to measure gene expression. NPY showed approximately 2-fold increases in their mRNA levels while sbGnRH showed 3-fold increases in adult fish. These results suggest that these peptides could be involved on hypothalamic regulation of Brazilian flounder sexual maturation.

scholarly journals Genome-Wide Analysis of Glucocorticoid-Responsive Transcripts in the Hypothalamic Paraventricular Region of Male Rats

Endocrinology ◽  
2018 ◽  
Vol 160 (1) ◽  
pp. 38-54 ◽  
Author(s):  
Keiichi Itoi ◽  
Ikuko Motoike ◽  
Ying Liu ◽  
Sam Clokie ◽  
Yasumasa Iwasaki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Receptor Gene ◽  
Male Rats ◽  
Regulatory Mechanisms ◽  
High Dose ◽  
Sequencing Analysis ◽  
Reverse Transcription Pcr ◽  
Genome Wide ◽  
A Genome

Abstract Glucocorticoids (GCs) are essential for stress adaptation, acting centrally and in the periphery. Corticotropin-releasing factor (CRF), a major regulator of adrenal GC synthesis, is produced in the paraventricular nucleus of the hypothalamus (PVH), which contains multiple neuroendocrine and preautonomic neurons. GCs may be involved in diverse regulatory mechanisms in the PVH, but the target genes of GCs are largely unexplored except for the CRF gene (Crh), a well-known target for GC negative feedback. Using a genome-wide RNA-sequencing analysis, we identified transcripts that changed in response to either high-dose corticosterone (Cort) exposure for 12 days (12-day high Cort), corticoid deprivation for 7 days (7-day ADX), or acute Cort administration. Among others, canonical GC target genes were upregulated prominently by 12-day high Cort. Crh was upregulated or downregulated most prominently by either 7-day ADX or 12-day high Cort, emphasizing the recognized feedback effects of GC on the hypothalamic-pituitary-adrenal (HPA) axis. Concomitant changes in vasopressin and apelin receptor gene expression are likely to contribute to HPA repression. In keeping with the pleotropic cellular actions of GCs, 7-day ADX downregulated numerous genes of a broad functional spectrum. The transcriptome response signature differed markedly between acute Cort injection and 12-day high Cort. Remarkably, six immediate early genes were upregulated 1 hour after Cort injection, which was confirmed by quantitative reverse transcription PCR and semiquantitative in situ hybridization. This study may provide a useful database for studying the regulatory mechanisms of GC-dependent gene expression and repression in the PVH.

Stress Hormone System and Epigenetics in Depression

2017 ◽  
Vol 41 (S1) ◽  
pp. S19-S19
Author(s):  
V. O’Keane ◽  
C. Farrell ◽  
K. Doolin ◽  
J. Chai ◽  
N. O’leary ◽  
...  
Keyword(s):  
Hpa Axis ◽  
Target Genes ◽  
Receptor Gene ◽  
Cell Receptor ◽  
Mrna Levels ◽  
Early Life Adversity ◽  
Generational Transmission ◽  
T Cell Receptor Gene ◽  
Competing Interest ◽  
Hormone System

BackgroundExposure to early life adversity (ELA) has been identified as a major risk factor in the development of major depressive disorder (MDD). It is hypothesized that a mediating mechanism may be environmentally induced alterations in gene function. In our REDEEM (Research in depression: endocrinology, epigenetics and neuroimaging) project we are examining possible epigenetic difference in some previously investigated target genes relevant to depression. To this end, methylation of the following genes were measured: NR3C1 (HPA axis), SLC6A4 (serotonin neurotransmitter function), and CD3ɛ (T cell receptor gene). We also looked at possible trans-generational transmission of epigenetic markers in a mother-baby sample.MethodsDNA was isolated from depressed patients and controls and babies and a portion of the above genes, encompassing our regions of interest, were amplified by PCR. Percentage methylation levels were measured by pyrosequencing. mRNA was also measured for some gene products to see if function was related to methylation. HPA axis function was measured with serial saliva samples throughout the day.Resultsto date: Methylation was increased in the CD3ɛ promoter in depressed subjects relative to controls. In the total group, those exposed to ELA had significantly increased methylation at this site. Levels of CD3ɛ mRNA levels were inversely related to methylation. There were some relationships between maternal ELA and baby methylation at the sites examined.ConclusionsConsistent with an allostatic model of ELA damage, our findings suggest an alteration in epigenetic function in acquired immunity and the HPA axis, mediated by ELA. Findings will be discussed.Disclosure of interestThe authors have not supplied their declaration of competing interest.

scholarly journals A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas)

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3840 ◽  
Author(s):  
Ming-An Tsai ◽  
I-Hua Chen ◽  
Jiann-Hsiung Wang ◽  
Shih-Jen Chou ◽  
Tsung-Hsien Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune System ◽  
Reference Genes ◽  
Animal Health ◽  
Rna Extraction ◽  
Disease Onset ◽  
Delphinapterus Leucas ◽  
Amplification Efficiency ◽  
Beluga Whales ◽  
Qrt Pcr

Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales (Delphinapterus leucas) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

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

2019 ◽  
Vol 4 ◽  
pp. 150 ◽  
Author(s):  
Antje K. Grotz ◽  
Fernando Abaitua ◽  
Elena Navarro-Guerrero ◽  
Benoit Hastoy ◽  
Daniel Ebner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Beta Cell ◽  
Er Stress ◽  
Cell Line ◽  
Cell Lines ◽  
Target Genes ◽  
Gene Knockout ◽  
Mrna Levels ◽  
Beta Cell Line ◽  
Human Beta Cell

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.

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