scholarly journals Evaluation of CRISPR gene-editing tools in zebrafish

BMC Genomics ◽  
2022 ◽  
Vol 23 (1) ◽  
Author(s):  
José M. Uribe-Salazar ◽  
Gulhan Kaya ◽  
Aadithya Sekar ◽  
KaeChandra Weyenberg ◽  
Cole Ingamells ◽  
...  
Keyword(s):  
Differentially Expressed Genes ◽  
Gene Editing ◽  
Differentially Expressed ◽  
Transient Effects ◽  
Cytoskeleton Organization ◽  
Functional Studies ◽  
Network Analyses ◽  
Molecular Features

Abstract Background Zebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create ‘knockout’ models. In particular, the use of G0 mosaic mutants has potential to increase throughput of functional studies significantly but may suffer from transient effects of introducing Cas9 via microinjection. Further, a large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods leaving uncertainty in choosing an optimal approach for zebrafish studies. Methods To systematically assess accuracy of tool predictions of on- and off-target gene editing, we subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes. We also investigate potential confounders of G0-based CRISPR screens by assaying control embryos for spurious mutations and altered gene expression. Results We compared our experimental in vivo editing efficiencies in mosaic G0 embryos with those predicted by eight commonly used gRNA design tools and found large discrepancies between methods. Assessing off-target mutations (predicted in silico and in vitro) found that the majority of tested loci had low in vivo frequencies (< 1%). To characterize if commonly used ‘mock’ CRISPR controls (larvae injected with Cas9 enzyme or mRNA with no gRNA) exhibited spurious molecular features that might exacerbate studies of G0 mosaic CRISPR knockout fish, we generated an RNA-seq dataset of various control larvae at 5 days post fertilization. While we found no evidence of spontaneous somatic mutations of injected larvae, we did identify several hundred differentially-expressed genes with high variability between injection types. Network analyses of shared differentially-expressed genes in the ‘mock’ injected larvae implicated a number of key regulators of common metabolic pathways, and gene-ontology analysis revealed connections with response to wounding and cytoskeleton organization, highlighting a potentially lasting effect from the microinjection process that requires further investigation. Conclusion Overall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.

scholarly journals Differential lncRNA/mRNA Expression Profiling and Functional Network Analyses in Bmp2 Deletion of Mouse Dental Papilla Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Feng Wang ◽  
Ran Tao ◽  
Li Zhao ◽  
Xin-Hui Hao ◽  
Yi Zou ◽  
...  
Keyword(s):  
Differentially Expressed Genes ◽  
Differentially Expressed ◽  
Knock Out ◽  
Protein Protein Interaction ◽  
Functional Studies ◽  
Network Analyses ◽  
Dental Papilla ◽  
Non Coding Rna ◽  
Mrna Expression Profiling ◽  
Dental Papilla Cells

Bmp2 is essential for dentin development and formation. Bmp2 conditional knock-out (KO) mice display a similar tooth phenotype of dentinogenesis imperfecta (DGI). To elucidate a foundation for subsequent functional studies of cross talk between mRNAs and lncRNAs in Bmp2-mediated dentinogenesis, we investigated the profiling of lncRNAs and mRNAs using immortalized mouse dental Bmp2 flox/flox (iBmp2fx/fx) and Bmp2 knock-out (iBmp2ko/ko) papilla cells. RNA sequencing was implemented to study the expression of the lncRNAs and mRNAs. Quantitative real-time PCR (RT-qPCR) was used to validate expressions of lncRNAs and mRNAs. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to predict functions of differentially expressed genes (DEGs). Protein–protein interaction (PPI) and lncRNA–mRNA co-expression network were analyzed by using bioinformatics methods. As a result, a total of 22 differentially expressed lncRNAs (16 downregulated vs 6 upregulated) and 227 differentially expressed mRNAs (133 downregulated vs. 94 upregulated) were identified in the iBmp2ko/ko cells compared with those of the iBmp2fx/fx cells. RT-qPCR results showed significantly differential expressions of several lncRNAs and mRNAs which were consistent with the RNA-seq data. GO and KEGG analyses showed differentially expressed genes were closely related to cell differentiation, transcriptional regulation, and developmentally relevant signaling pathways. Moreover, network-based bioinformatics analysis depicted the co-expression network between lncRNAs and mRNAs regulated by Bmp2 in mouse dental papilla cells and symmetrically analyzed the effect of Bmp2 during dentinogenesis via coding and non-coding RNA signaling.

6 EXPRESSION PROFILING OF SINGLE BOVINE EMBRYOS REVEALS SIGNIFICANT EFFECTS OF IN VITRO MATURATION, FERTILIZATION AND CULTURE

2006 ◽  
Vol 18 (2) ◽  
pp. 111
Author(s):  
S. L. Smith ◽  
L.-Y. Sung ◽  
R. Page ◽  
B. Henderson ◽  
F. Du ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differentially Expressed Genes ◽  
Oocyte Maturation ◽  
Expression Profiling ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Differentially Expressed ◽  
In Vitro Oocyte Maturation

Cattle and sheep embryos transferred after in vitro production are often afflicted by large offspring syndrome (LOS), which has been correlated with the presence of serum and/or cell co-culture. Previous research indicates that post-fertilization culture affects blastocyst quality and gene expression, and in vitro oocyte maturation and fertilization impact developmental competence. To dissect the effects of in vitro maturation, fertilization, and culture, we compared the expression profiles of single bovine blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF, n = 15); (2) in vivo maturation, in vivo fertilization, and in vitro culture (IVD, n = 14); and (3) in vivo maturation, fertilization, and development (AI, n = 14). For in vitro culture, the embryos were cultured for 2 days in CR1aa medium with bovine serum albumin (BSA) and then transferred to CR1aa with 10% fetal bovine serum (FBS) with cumulus cells until Day 7, at which time the embryos were vitrified. IVD zygotes were surgically collected from two superovulated Holstein donor cows 24 h post-insemination and cultured in the same system. To conduct expression profiling, total RNA was isolated from individual thawed embryos. The RNA was subjected to three rounds of amplification utilizing a previously adapted and validated T7 linear amplification protocol. Amplified RNA from each embryo and from a standard reference was indirectly labeled with Cy3 or Cy5 by dye swap and hybridized to a custom bovine cDNA microarray containing ~6300 unique genes. After Loess normalization, an ANOVA model (GeneSpring 6.1 and SAS 9.0) was used to identify differentially expressed genes. The P-values were adjusted for multiple comparisons using the false discovery rate approach, and a e2-fold differential criterion was applied. A subset of the differentially expressed genes was verified by real-time RT-PCR. The blastocyst rates for IVF and IVD embryos were 37% and 75%, respectively. There were 305, 365, and 200 genes differentially expressed between the AI and IVD, the IVF and IVD, and the AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and the IVD embryos, making these potential candidates for LOS. There were 61 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology categories 'RNA processing' and 'RNA binding' were over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on embryonic gene expression. This work was supported by USDA grants to X.Y., H.A.L., and X.C.T.

scholarly journals Tumor-suppressive function and mechanism of HOXB13 in right-sided colon cancer

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Binbin Xie ◽  
Bingjun Bai ◽  
Yuzi Xu ◽  
Yunlong Liu ◽  
Yiming Lv ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Tissue Sample ◽  
Suppressive Effect ◽  
Gene Expression Omnibus ◽  
Differentially Expressed ◽  
Antitumor Effects ◽  
Molecular Features ◽  
Induced Apoptosis

AbstractRight-sided colon cancer (RCC) and left-sided colon cancer (LCC) differ in their clinical and molecular features. An investigation of differentially expressed genes (DEGs) between RCC and LCC could contribute to targeted therapy for colon cancer, especially RCC, which has a poor prognosis. Here, we identified HOXB13, which was significantly less expressed in RCC than in LCC and associated with prognosis in RCC, by using 5 datasets from the Gene Expression Omnibus (GEO). Tissue sample analysis showed that HOXB13 was differentially expressed between normal and only RCC tumor tissues. HOXB13 inhibited colon cancer cell proliferation and induced apoptosis both in vitro and in vivo. Furthermore, we found that HOXB13 might be regulated by DNMT3B and suppress C-myc expression to exert antitumor effects via β-catenin/TCF4 signals in RCC. In conclusion, the current study is the first to demonstrate that HOXB13 has a tumor-suppressive effect in RCC. High expression levels of HOXB13 are associated with prolonged overall survival in patients with RCC. The DNMT3B-HOXB13-C-myc signaling axis might be a molecular target for the treatment of RCC.

scholarly journals The targeted inhibition of prostate cancer by iron-based nanoparticles based on bioinformatics

2020 ◽  
pp. 088532822097524
Author(s):  
Feng Xiao ◽  
Jiayu Liu ◽  
Yongbo Zheng ◽  
Zhen Quan ◽  
Wei Sun ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Gene Therapy ◽  
Cancer Cells ◽  
Differentially Expressed Genes ◽  
Differentially Expressed ◽  
Urogenital System ◽  
Prostate Cancer Cells ◽  
Iron Based

Prostate cancer is an epithelial malignant tumor of the prostate, and it is one of the malignant tumors with a high incidence of urogenital system in men. The local treatment of prostate cancer is mainly radical resection and radical radiotherapy, but they are not applicable to advanced prostate cancer. Systemic therapy mainly includes targeted therapy and immunotherapy which could cause many complications, and will affect the prognosis and quality of life of patients. It is urgent to find new treatments for prostate cancer. Bioinformatics offers hope for us to find reliable therapeutic targets. Bioinformatics can use the tumor informations in database and analyze them to screen out the best differentially expressed genes. Using the selected differentially expressed genes as targets, a gene interference plasmid was designed, and the constructed plasmid was used for targeted gene therapy. There are some problems about gene therapy that need to be solved, such as how to transfer genes to target cells is also an important challenge. Due to their large molecular weight and hydrophilic nature, they cannot enter cells through passive diffusion mechanisms. Here we synthesized a DNA carrier used surface modified iron based nanoparticles, and used it to load plasmid including ShRNA which can inhibit the expression of oncogene SLC4A4 selected by bioinformatics’ method. After that we use this iron based nanoparticles/plasmid DNA nanocomposite to treat prostate cancer cells in vitro and in vivo. The target gene SLC4A4 we had selected using bioinformatics had a strong effect on the proliferation of prostate cells; Our nanocomposite could inhibit the expression of SLC4A4 effectively, it had strong inhibitory effects on prostate cancer cells both in vivo and in vitro, and can be used as a potential method for prostate cancer treatment.

scholarly journals Genome-Wide Expression Analysis of Burkholderia pseudomallei Infection ina Hamster Model of Acute Melioidosis

2006 ◽  
Vol 74 (10) ◽  
pp. 5465-5476 ◽  
Author(s):  
Apichai Tuanyok ◽  
Marina Tom ◽  
John Dunbar ◽  
Donald E. Woods
Keyword(s):  
Gene Expression ◽  
Differentially Expressed Genes ◽  
Energy Production ◽  
Burkholderia Pseudomallei ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Bacterial Growth Rate ◽  
Hamster Model

ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In the current studies we have examined gene expression in B. pseudomallei in an animal model of acute melioidosis using whole-genome microarrays. Gene expression profiles were generated by comparing transcriptional levels of B. pseudomallei-expressed genes in infected hamster organs including liver, lung, and spleen following intraperitoneal and intranasal routes of infection to those from bacteria grown in vitro. Differentially expressed genes were similar in infected livers irrespective of the route of infection. Reduced expression of a number of housekeeping genes suggested a lower bacterial growth rate during infection. Energy production during growth in vivo involved specific biochemical pathways such as isomerization of 3-phosphoglycerate, catabolism of d-glucosamine and inositol, and biosynthesis of particular amino acids. In addition, the induction of genes known to be involved in oxidative phosphorylation including ubiquinol oxidase, ferredoxin oxidoreductase, and formate dehydrogenase enzymes suggested the use of alternative pathways for energy production, while the expression of genes coding for ATP-synthase and NADH-dehydrogenase enzymes was reduced. Our studies have identified differentially expressed genes which include potential virulence genes such as those for a putative phospholipase C and a putative two-component regulatory system, and they have also provided a better understanding of bacterial metabolism in response to the host environment during acute melioidosis.

scholarly journals 216 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN BOVINE EMBRYOS CULTURED IN VIVO OR IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 259 ◽  
Author(s):  
D. Corcoran ◽  
T. Fair ◽  
D. Rizos ◽  
G.W. Smith ◽  
P.M. Coussens ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Differentially Expressed Genes ◽  
Intracellular Signalling ◽  
Differentially Expressed ◽  
Regulation Of Transcription ◽  
Gene Transcript ◽  
Bovine Embryo ◽  
Total Rna

The post-fertilization culture environment of the bovine embryo is known to influence the quality of the resulting blastocyst, manifested in terms of morphology, cryotolerance, and the relative gene transcript abundance of several candidate genes. This may have consequences for the pregnancy rate following embryo transfer. The objective of the current study was to take a broader approach toward identifying differentially expressed genes in bovine blastocysts derived from either in vivo or in vitro culture. Presumptive zygotes, produced by in vitro maturation and fertilization, were randomly assigned to one of two groups and cultured for 6 days, either in vitro in SOF medium, or in vivo in the ewe oviduct following transfer by mid-ventral laparotomy. Blastocysts were recovered from both systems on Day 7 after insemination, snap frozen in liquid nitrogen, and stored at −80°C. Total RNA was extracted from 50 blastocysts for each culture group from each of four replicates using the PicoPureTM RNA Isolation Kit (ARCTURUS, Mountain View, CA 94043, USA). The RiboAmpTM RNA Amplification Kit (ARCTURUS) was used to linearly amplify the mRNA fraction of total RNA using double-stranded cDNA as template in a T7 RNA polymerase-catalyzed amplification. Samples from both culture environments were differentially labelled using N-hydroxysuccinimide (NHS)-activated fluorescent Cy3 or Cy5 dyes (Amersham Pharmacia Ltd., Piscataway, NJ, USA) and were hybridized onto a cDNA microarray. Each microarray contained 3888 total spots, with 932 bovine EST clone inserts developed from a normalized bovine total leukocyte (BOTL) cDNA library and an additional 459 amplicons representing additional genes including cytokines, receptors, signal transduction molecules, transcription and growth factors, enzymes, cell cycle regulators, and cellular components. Data were normalized and an expression ratio calculated between the two groups. This was compared to 1 within each of the 4 replicates by Student's t-test. Microarray analysis identified 15 gene transcripts that were differentially expressed P ≤ 0.05) between blastocysts produced in vivo or in vitro. Among these, four genes involved in transcription (nuclear receptor co-repressor 1, zinc finger protein 22, CCR4-NOT transcription complex, DOT1) and two genes involved in intracellular signalling (proteasome 26S subunit non-ATPase 13 and guanine nucleotide binding protein) had a higher mRNA expression level in blastocysts produced in vivo compared to those produced in vitro. In addition, Connexin 43, a gene involved in gap junction formation, was down regulated following in vitro culture which is consistent with our previous studies. Among the genes up-regulated following in vitro culture were WNT2B wingless-type MMTV integration site, CD103 integrin, and tumor necrosis factor superfamily member 8. In conclusion, we have identified previously uncharacterized, differentially expressed genes involved in cell communication, intracellular signalling, and regulation of transcription in bovine blastocysts cultured in vivo or in vitro. This work was supported by Science Foundation Ireland under Grant No. 02/IN1/B78.

scholarly journals CircFAM13B promotes the proliferation of hepatocellular carcinoma by sponging miR-212, upregulating E2F5 expression and activating the P53 pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Ying Xie ◽  
Xiaofeng Hang ◽  
Wensheng Xu ◽  
Jing Gu ◽  
Yuanjing Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatocellular Carcinoma ◽  
Gene Expression Omnibus ◽  
Differentially Expressed ◽  
In Vivo Studies ◽  
Circular Rnas ◽  
Novel Target ◽  
Underlying Mechanisms

Abstract Background Most of the biological functions of circular RNAs (circRNAs) and the potential underlying mechanisms in hepatocellular carcinoma (HCC) have not yet been discovered. Methods In this study, using circRNA expression data from HCC tumor tissues and adjacent tissues from the Gene Expression Omnibus database, we identified out differentially expressed circRNAs and verified them by qRT-PCT. Functional experiments were performed to evaluate the effects of circFAM13B in HCC in vitro and in vivo. Results We found that circFAM13B was the most significantly differentially expressed circRNA in HCC tissue. Subsequently, in vitro and in vivo studies also demonstrated that circFAM13B promoted the proliferation of HCC. Further studies revealed that circFAM13B, a sponge of miR-212, is involved in the regulation of E2F5 gene expression by competitively binding to miR-212, inhibits the activation of the P53 signalling pathway, and promotes the proliferation of HCC cells. Conclusions Our findings revealed the mechanism underlying the regulatory role played by circFAM13B, miR-212 and E2F5 in HCC. This study provides a new theoretical basis and novel target for the clinical prevention and treatment of HCC.

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