scholarly journals Macrophage Modification Strategies for Efficient Cell Therapy

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1535 ◽  
Author(s):  
Anastasiya S. Poltavets ◽  
Polina A. Vishnyakova ◽  
Andrey V. Elchaninov ◽  
Gennady T. Sukhikh ◽  
Timur Kh. Fatkhudinov
Keyword(s):  
Gene Expression ◽  
Gene Editing ◽  
Target Selection ◽  
Model Systems ◽  
Attractive Therapeutic Target ◽  
Specific Alteration ◽  
Inflammatory Phenotypes ◽  
Vector Delivery

Macrophages, important cells of innate immunity, are known for their phagocytic activity, capability for antigen presentation, and flexible phenotypes. Macrophages are found in all tissues and therefore represent an attractive therapeutic target for the treatment of diseases of various etiology. Genetic programming of macrophages is an important issue of modern molecular and cellular medicine. The controllable activation of macrophages towards desirable phenotypes in vivo and in vitro will provide effective treatments for a number of inflammatory and proliferative diseases. This review is focused on the methods for specific alteration of gene expression in macrophages, including the controllable promotion of the desired M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes in certain pathologies or model systems. Here we review the strategies of target selection, the methods of vector delivery, and the gene editing approaches used for modification of macrophages.

scholarly journals Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in ‘mock’ control embryos

2020 ◽  
Author(s):  
José M. Uribe-Salazar ◽  
Aadithya Sekar ◽  
Gulhan Kaya ◽  
KaeChandra Weyenberg ◽  
Cole Ingamells ◽  
...  
Keyword(s):  
Gene Editing ◽  
Functional Characterization ◽  
Negative Control ◽  
Zebrafish Genome ◽  
High Variability ◽  
Model Systems ◽  
Segmental Duplications ◽  
Mock Control

ABSTRACTZebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create ‘knockout’ models. Due to the large number of available tools to design CRISPR assays and diversity of theories/model systems they were originally built on, we sought to systematically compare computational and empirical approaches for predicting gene-editing efficacy in zebrafish. We subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes and assayed individual editing efficiencies. We compared our experimental in vivo efficiencies in mosaic G0 embryos with those predicted by seven 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%). Moreover, understanding that recent segmental duplications in the zebrafish genome could exacerbate CRISPR targeting of individual genes, we cataloged these loci and have made them available as a resource. Lastly, we assessed the transcriptome of negative ‘mock’ control CRISPR larvae injected with Cas9 enzyme or mRNA with no gRNA using RNA-seq and identified differentially expressed genes with high variability between injections. Using these same data, we discovered on average ~60 putative somatic mosaic frameshift mutations impacting genes per pool of injected larvae, potentially due to background cutting of DNA with Cas9 in the absence of gRNA. To verify this previously unreported phenomenon in zebrafish, we validated seven of twelve genes tested carrying low frequency mosaic somatic mutations in the genomes of a separate batch of injected larvae. These results suggest that negative control embryos may carry mutations within genes leading to spurious phenotypes. Overall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.AUTHOR SUMMARYZebrafish have proven to be a powerful model organism for the functional characterization of genes. Development of new workflows targeting individual or multiple genes simultaneously require a thorough understanding of the advantages and limitations of current available methods for CRISPR-editing in zebrafish. Here, we systematically evaluated on- and off-target efficiencies prediction methods of 50 gRNAs by experimentally testing their CRISPR cutting efficiencies in embryos. Moreover, we performed a global assessment of duplicated portions of the zebrafish genome, providing a powerful resource for the design of future CRISPR assays. Lastly, we evaluated the possibility that spurious editing occurs in samples injected with the Cas9 nuclease without a gRNA, which are commonly used as a baseline control. This analysis revealed high variability in gene expression and the presence of frameshift variants in larvae injected solely with Cas9, suggesting that additional caution should be taken when using these samples as baseline controls in functional characterizations of genes.

scholarly journals A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wen-Hsin Liu ◽  
Kerstin Völse ◽  
Daniela Senft ◽  
Irmela Jeremias
Keyword(s):  
Protein Function ◽  
Gene Editing ◽  
Precursor Cell ◽  
Model Systems ◽  
Leukemia Cells ◽  
Reporter System ◽  
Dual Reporter ◽  
Primary Leukemia

AbstractCRISPR/Cas9 represents a valuable tool to determine protein function, but technical hurdles limit its use in challenging settings such as cells unable to grow in vitro like primary leukemia cells and xenografts derived thereof (PDX). To enrich CRISPR/Cas9-edited cells, we improved a dual-reporter system and cloned the genomic target sequences of the gene of interest (GOI) upstream of an out-of-frame fluorochrome which was expressed only upon successful gene editing. To reduce rounds of in vivo passaging required for PDX leukemia growth, targets of 17 GOI were cloned in a row, flanked by an improved linker, and PDX cells were lentivirally transduced for stable expression. The reporter enriched scarce, successfully gene-edited PDX cells as high as 80%. Using the reporter, we show that KO of the SRC-family kinase LYN increased the response of PDX cells of B precursor cell ALL towards Vincristine, even upon heterozygous KO, indicating haploinsufficiency. In summary, our reporter system enables enriching KO cells in technically challenging settings and extends the use of gene editing to highly patient-related model systems.

Effect of X-irradiation on gene expression of rat liver chemokines: in vivo and in vitro studies

2008 ◽  
Vol 46 (01) ◽  
Author(s):  
F Moriconi ◽  
H Christiansen ◽  
H Christiansen ◽  
N Sheikh ◽  
J Dudas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rat Liver ◽  
In Vitro Studies ◽  
X Irradiation

Vibrio harveyi virulence gene expression in vitro and in vivo during infection in black tiger shrimp Penaeus monodon

2020 ◽  
Vol 139 ◽  
pp. 153-160
Author(s):  
S Peeralil ◽  
TC Joseph ◽  
V Murugadas ◽  
PG Akhilnath ◽  
VN Sreejith ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virulence Genes ◽  
Vibrio Harveyi ◽  
Penaeus Monodon ◽  
Challenge Test ◽  
Virulence Gene ◽  
Economic Losses ◽  
Virulence Gene Expression

Luminescent Vibrio harveyi is common in sea and estuarine waters. It produces several virulence factors and negatively affects larval penaeid shrimp in hatcheries, resulting in severe economic losses to shrimp aquaculture. Although V. harveyi is an important pathogen of shrimp, its pathogenicity mechanisms have yet to be completely elucidated. In the present study, isolates of V. harveyi were isolated and characterized from diseased Penaeus monodon postlarvae from hatcheries in Kerala, India, from September to December 2016. All 23 tested isolates were positive for lipase, phospholipase, caseinase, gelatinase and chitinase activity, and 3 of the isolates (MFB32, MFB71 and MFB68) showed potential for significant biofilm formation. Based on the presence of virulence genes, the isolates of V. harveyi were grouped into 6 genotypes, predominated by vhpA+ flaB+ ser+ vhh1- luxR+ vopD- vcrD+ vscN-. One isolate from each genotype was randomly selected for in vivo virulence experiments, and the LD50 ranged from 1.7 ± 0.5 × 103 to 4.1 ± 0.1 × 105 CFU ml-1. The expression of genes during the infection in postlarvae was high in 2 of the isolates (MFB12 and MFB32), consistent with the result of the challenge test. However, in MFB19, even though all genes tested were present, their expression level was very low and likely contributed to its lack of virulence. Because of the significant variation in gene expression, the presence of virulence genes alone cannot be used as a marker for pathogenicity of V. harveyi.

Evidence that gene expression of ovarian follicular tight junction proteins is regulated in vivo and in vitro in cattle

2017 ◽  
Vol 95 (3) ◽  
pp. 1313 ◽  
Author(s):  
L. Zhang ◽  
L. F. Schütz ◽  
C. L. Robinson ◽  
M. L. Totty ◽  
L. J. Spicer
Keyword(s):  
Gene Expression ◽  
Tight Junction ◽  
Tight Junction Proteins ◽  
Junction Proteins

Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

2005 ◽  
Vol 83 (4) ◽  
pp. 535-547 ◽  
Author(s):  
Gareth N Corry ◽  
D Alan Underhill
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Current Knowledge ◽  
Nuclear Architecture ◽  
Subnuclear Localization ◽  
Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

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