scholarly journals Inactivation of porcine interleukin-1β results in failure of rapid conceptus elongation

2017 ◽  
Vol 115 (2) ◽  
pp. 307-312 ◽  
Author(s):  
Jeffrey J. Whyte ◽  
Ashley E. Meyer ◽  
Lee D. Spate ◽  
Joshua A. Benne ◽  
Raissa Cecil ◽  
...  
Keyword(s):  
Gene Editing ◽  
Mammalian Species ◽  
Interleukin 1Β ◽  
Corpora Lutea ◽  
Morphological Transition ◽  
Wild Type ◽  
Morphological Transformation ◽  
Proinflammatory Response ◽  
Knock Out ◽  
Estrogen Synthesis

Conceptus expansion throughout the uterus of mammalian species with a noninvasive epitheliochorial type of placentation is critical establishing an adequate uterine surface area for nutrient support during gestation. Pig conceptuses undergo a unique rapid morphological transformation to elongate into filamentous threads within 1 h, which provides the uterine surface to support development and maintain functional corpora lutea through the production of estrogen. Conceptus production of a unique interleukin 1β, IL1B2, temporally increases during the period of trophoblast remodeling during elongation. CRISPR/Cas9 gene editing was used to knock out pig conceptus IL1B2 expression and the secretion of IL1B2 during the time of conceptus elongation. Trophoblast elongation occurred on day 14 in wild-type (IL1B2+/+) conceptuses but did not occur in ILB2-null (IL1B2−/−) conceptuses. Although the morphological transition of IL1B2−/− conceptuses was inhibited, expression of a number of conceptus developmental genes was not altered. However, conceptus aromatase expression and estrogen secretion were decreased, indicating that IL1B2 may be involved in the spatiotemporal increase in conceptus estrogen synthesis needed for the establishment of pregnancy in the pig and may serve to regulate the proinflammatory response of endometrium to IL1B2 during conceptus elongation and attachment to the uterine surface.

scholarly journals New insights into the construction of wild-type Saba pig-derived Escherichia coli irp2 gene deletion strains

3 Biotech ◽  
2021 ◽  
Vol 11 (9) ◽  
Author(s):  
Bo Zhang ◽  
Hongdan Wang ◽  
Weiwei Zhao ◽  
Chunlan Shan ◽  
Chaoying Liu ◽  
...  
Keyword(s):  
Iron Uptake ◽  
Gene Editing ◽  
Gene Deletion ◽  
Growth Characteristics ◽  
Wild Type ◽  
Knock Out ◽  
E Coli ◽  
Functional Studies ◽  
Key Points ◽  
Type Strains

AbstractTo construct wild-type E. coli irp2 gene deletion strains, CRISPR/Cas9 gene editing technology was used, and the difficulty and key points of gene editing of wild-type strains were analyzed. Based on the resistance of the CRISPR/Cas9 system expression vector, 4 strains of 41 E. coli strains isolated from Saba pigs were selected as the target strains for the deletion of the irp2 gene, which were sensitive to both ampicillin and kanamycin. Then, CRISPR/Cas9 technology was combined with homologous recombination technology to construct recombinant vectors containing Cas9, sgRNA and donor sequences to knock out the irp2 gene. Finally, the absence of the irp2 gene in E. coli was further verified by iron uptake assays, iron carrier production assays and growth curve measurements. The results showed that three of the selected strains showed single base mutations and deletions (Δirp2-1, Δirp2-2 and Δirp2-3). The deletion of the irp2 gene reduced the ability of E. coli to take up iron ions and produce iron carriers, but not affect the growth characteristics of E. coli. It is shown that the CRISPR/Cas9 knock-out system constructed in this study can successfully knock out the irp2 gene of the wild-type E. coli. Our results providing new insights into genome editing in wild-type strains, which enable further functional studies of the irp2 gene in wild-type E. coli.

scholarly journals Enhanced soluble sugar content in tomato fruit using CRISPR/Cas9-mediated SlINVINH1 and SlVPE5 gene editing

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12478
Author(s):  
Baike Wang ◽  
Ning Li ◽  
Shaoyong Huang ◽  
Jiahui Hu ◽  
Qiang Wang ◽  
...  
Keyword(s):  
Gene Editing ◽  
Single Gene ◽  
Tomato Fruit ◽  
Sugar Content ◽  
Soluble Sugar ◽  
Wild Type ◽  
Tomato Fruits ◽  
Knock Out ◽  
Soluble Sugar Content ◽  
Soluble Solid

Soluble sugar is known to improve the sweetness and increase tomato sauce yield. Studies have focused on improving the content of soluble sugar in tomato fruits, usually by promoting functional genes. We studied two genes (SlINVINH1 and SlVPE5) that inhibited the accumulation of soluble sugar in tomato fruits and obtained two genes’ knocked-out lines (CRISPR-invinh1 or CRISPR-vpe5) using CRISPR/Cas9. Aggregated lines with CRISPR-invinh1 and CRISPR-vpe5 were gained by hybridization and self-pollination. Compared to wild-type lines, the glucose, fructose, and total soluble solid (TSS) contents of CRISPR-invinh1 and CRISPR-vpe5 increased significantly. Glucose, fructose, and TSS levels further improved simultaneously with CRISPR-invinh1 and CRISPR-vpe5 than with single gene knock-out lines. This indicates that these genes have a synergistic effect and will increase the soluble sugar content. Thus, the knock-out SlINVINH1 and SlVPE5 may provide a practical basis for improving the sweetness of tomato fruits and their processing quality.

scholarly journals Effective MSTN Gene Knockout by AdV-Delivered CRISPR/Cas9 in Postnatal Chick Leg Muscle

2020 ◽  
Vol 21 (7) ◽  
pp. 2584 ◽  
Author(s):  
Ke Xu ◽  
Cheng Xiao Han ◽  
Hao Zhou ◽  
Jin Mei Ding ◽  
Zhong Xu ◽  
...  
Keyword(s):  
Growth And Development ◽  
Gene Editing ◽  
Gene Knockout ◽  
Muscle Growth ◽  
Meat Production ◽  
Wild Type ◽  
Knock Out ◽  
Mstn Gene ◽  
Differentiation And Proliferation ◽  
Transcriptomic Changes

Muscle growth and development are important aspects of chicken meat production, but the underlying regulatory mechanisms remain unclear and need further exploration. CRISPR has been used for gene editing to study gene function in mice, but less has been done in chick muscles. To verify whether postnatal gene editing could be achieved in chick muscles and determine the transcriptomic changes, we knocked out Myostatin (MSTN), a potential inhibitor of muscle growth and development, in chicks and performed transcriptome analysis on knock-out (KO) muscles and wild-type (WT) muscles at two post-natal days: 3d (3-day-old) and 14d (14-day-old). Large fragment deletions of MSTN (>5 kb) were achieved in all KO muscles, and the MSTN gene expression was significantly downregulated at 14d. The transcriptomic results indicated the presence of 1339 differentially expressed genes (DEGs) between the 3d KO and 3d WT muscles, as well as 597 DEGs between 14d KO and 14d WT muscles. Many DEGs were found to be related to cell differentiation and proliferation, muscle growth and energy metabolism. This method provides a potential means of postnatal gene editing in chicks, and the results presented here could provide a basis for further investigation of the mechanisms involved in muscle growth and development.

Functional characterization of the H+/K+ ATPase in wild type and gastrin knock-out mice

2007 ◽  
Vol 45 (05) ◽  
Author(s):  
A Schnur ◽  
P Hegyi ◽  
V Venglovecz ◽  
Z Rakonczay ◽  
I Ignáth ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Wild Type ◽  
Knock Out ◽  
Knock Out Mice

Pharmaceutical and Biotechnological Applications of Transgenic Technology

2018 ◽  
Vol 11 (4) ◽  
pp. 4145-4153
Author(s):  
D Samba Reddy ◽  
Tina Reddy
Keyword(s):  
Mammalian Species ◽  
Genetic Diseases ◽  
Transgenic Animals ◽  
Transgenic Animal ◽  
Pharmaceutical Products ◽  
Conditional Knockout ◽  
Knock Out ◽  
Transgenic Lines ◽  
Health And Disease ◽  
Or Genes

A transgenic animal is a genetically modified species in which researchers have modified an existing gene or genes by genetic engineering techniques. Genetic modification involves the mutation, insertion, or deletion of genes. Mouse is the most widely used mammalian species for creating transgenic lines. There are two types of transgenic animals: (i) gene deleted (“knock-out”) and (ii) gene overexpressed (“knock-in”). The loss or gain of gene activity often causes changes in a mouse's phenotype, which includes appearance, behavior and other observable characteristics. Knockout mice are key animal models for studying the role of genes which have been sequenced but whose functions have not been determined.  They include constitutive knockouts (gene deleted since birth) and conditional knockout (gene turned off later after birth).  The first knockout mouse was created in 1989 by Mario Capecchi, Martin Evans, and Oliver Smithies, for which they were awarded the 2007 Nobel Prize in Physiology or Medicine.  Transgenic mouse models have revolutionized the biomedical research and provided a power tool for understanding health and disease. Transgenic animals have been created for bulk production of biotechnology and pharmaceutical products.  In 2009, the FDA approved the first human biological drug ATryn, an anticoagulant extracted from the transgenic goat's milk. The recently discovered CRISPER gene editing technology is providing new frontiers in correcting abnormal genes and hopefully provide cures for genetic diseases in the future.    

scholarly journals Effects of cofD gene knock-out on the methanogenesis of Methanobrevibacter ruminantium

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jian Ma ◽  
Xueying Wang ◽  
Ting Zhou ◽  
Rui Hu ◽  
Huawei Zou ◽  
...  
Keyword(s):  
Growth Rate ◽  
Specific Growth ◽  
Production Capacity ◽  
Maximum Amount ◽  
Logarithmic Phase ◽  
Wild Type ◽  
Knock Out ◽  
Maximum Biomass ◽  
Reproductive Ability ◽  
Methanobrevibacter Ruminantium

AbstractThis study aimed to investigate the effects of cofD gene knock-out on the synthesis of coenzyme F420 and production of methane in Methanobrevibacter ruminantium (M. ruminantium). The experiment successfully constructed a cofD gene knock-out M. ruminantium via homologous recombination technology. The results showed that the logarithmic phase of mutant M. ruminantium (12 h) was lower than the wild-type (24 h). The maximum biomass and specific growth rate of mutant M. ruminantium were significantly lower (P < 0.05) than those of wild-type, and the maximum biomass of mutant M. ruminantium was approximately half of the wild-type; meanwhile, the proliferation was reduced. The synthesis amount of coenzyme F420 of M. ruminantium was significantly decreased (P < 0.05) after the cofD gene knock-out. Moreover, the maximum amount of H2 consumed and CH4 produced by mutant were 14 and 2% of wild-type M. ruminantium respectively. In conclusion, cofD gene knock-out induced the decreased growth rate and reproductive ability of M. ruminantium. Subsequently, the synthesis of coenzyme F420 was decreased. Ultimately, the production capacity of CH4 in M. ruminantium was reduced. Our research provides evidence that cofD gene plays an indispensable role in the regulation of coenzyme F420 synthesis and CH4 production in M. ruminantium.

Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice

Development ◽  
1999 ◽  
Vol 126 (13) ◽  
pp. 3015-3025 ◽  
Author(s):  
G.H. Fong ◽  
L. Zhang ◽  
D.M. Bryce ◽  
J. Peng
Keyword(s):  
Endothelial Cells ◽  
Population Density ◽  
Cell Fate ◽  
Vascular System ◽  
Primitive Streak ◽  
Cellular Level ◽  
Wild Type ◽  
Primary Defect ◽  
Knock Out ◽  
Vascular Channels

We previously demonstrated the essential role of the flt-1 gene in regulating the development of the cardiovascular system. While the inactivation of the flt-1 gene leads to a very severe disorganization of the vascular system, the primary defect at the cellular level was unknown. Here we report a surprising finding that it is an increase in the number of endothelial progenitors that leads to the vascular disorganization in flt-1(−/−) mice. At the early primitive streak stage (prior to the formation of blood islands), hemangioblasts are formed much more abundantly in flt-1(−/−) embryos. This increase is primarily due to an alteration in cell fate determination among mesenchymal cells, rather than to increased proliferation, migration or reduced apoptosis of flt-1(−/−) hemangioblasts. We further show that the increased population density of hemangioblasts is responsible for the observed vascular disorganization, based on the following observations: (1) both flt-1(−/−) and flt-1(+/+) endothelial cells formed normal vascular channels in chimaeric embryos; (2) wild-type endothelial cells formed abnormal vascular channels when their population density was significantly increased; and (3) in the absence of wild-type endothelial cells, flt-1(−/−) endothelial cells alone could form normal vascular channels when sufficiently diluted in a developing embryo. These results define the primary defect in flt-1(−/−) embryos at the cellular level and demonstrate the importance of population density of progenitor cells in pattern formation.

TAMI-37. STEAROYL-COA DESATURASE 1 IS ESSENTIAL FOR THE GROWTH OF IDH MUTANT GLIOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi206-vi206
Author(s):  
Tomohiro Yamasaki ◽  
Lumin Zhang ◽  
Tyrone Dowdy ◽  
Adrian Lita ◽  
Mark Gilbert ◽  
...  
Keyword(s):  
Glioma Cell ◽  
De Novo ◽  
Glioma Cells ◽  
Model Systems ◽  
De Novo Lipogenesis ◽  
Wild Type ◽  
Knock Out ◽  
Stearoyl Coa Desaturase ◽  
Scd1 Expression

Abstract BACKGROUND Increased de novo lipogenesis is a hallmark of cancer metabolism. In this study, we interrogated the role of de novo lipogenesis in IDH1 mutated glioma’s growth and identified the key enzyme, Stearoyl-CoA desaturase 1 (SCD1) that provides this growth advantage. MATERIALS ANDMETHODS We prepared genetically engineered glioma cell lines (U251 wild-type: U251WT and U251 IDHR132H mutant: U251RH) and normal human astrocytes (empty vector induced-NHA: NHAEV and IDHR132H mutant: NHARH). Lipid metabolic analysis was conducted by using LC-MS and Raman imaging microscopy. SCD1 expression was investigated by The Cancer Genome Atlas (TCGA) data analysis and Western-blotting method. Knock-out of SCD1 was conducted by using CRISPR/Cas9 and shRNA. RESULTS Previously, we showed that IDH1 mut glioma cells have increased monounsaturated fatty acids (MUFAs). TCGA data revealed IDH mut glioma shows significantly higher SCD1 mRNA expression than wild-type glioma. Our model systems of IDH1 mut (U251RH, NHARH) showed increased expression of this enzyme compared with their wild-type counterpart. Moreover, addition of D-2HG to U251WT increased SCD1 expression. Herein, we showed that inhibition of SCD1 with CAY10566 decreased relative cell number and sphere forming capacity in a dose-dependent manner. Furthermore, addition of MUFAs were able to rescue the SCD1 inhibitor induced-cell death and sphere forming capacity. Knock out of SCD1 revealed decreased cell proliferation and sphere forming ability. Decreasing lipid content from the media did not alter the growth of these cells, suggesting that glioma cells rely on de novo lipid synthesis rather than scavenging them from the microenvironment. CONCLUSION Overexpression of IDH mutant gene altered lipid composition in U251 cells to enrich MUFA levels and we confirmed that D-2HG caused SCD1 upregulation in U251WT. We demonstrated the glioma cell growth requires SCD1 expression and the results of the present study may provide novel insights into the role of SCD1 in IDH mut gliomas growth.

CRISPR–Cas9 gene editing as a tool for developing immunotherapy for cancer

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
T Cells ◽  
Immune Cells ◽  
Peripheral Blood ◽  
Gene Editing ◽  
Genetically Engineered ◽  
Cancer Resistance ◽  
Knock Out ◽  
Tumor Tissues ◽  
Engineered T Cells ◽  
Long Time

T cells following genome editing and transformation might be detectable in peripheral blood and tumor tissues for a long time, even more than a year. The types and diversity of T-cells in peripheral blood and tumor tissues changed following transfusion of genetically modified T-cells, and some highly suspected T-cells targeting cancer cells grew, increasing the proportion of such cells. Moreover, after getting genetically engineered T cells, anticancer cytokine secretion increased. T cells changed by gene editing have certain functions, at least from an immunological standpoint. The first clinical research using the CRISPR–Cas9 gene editing method for cancer resistance is more complicated: Using CRISPR–Cas9 gene editing technology to concurrently knock out, amplify, activate and reinfuse three genes in human immune cells. This therapeutic strategy is more demanding, because the changed immune cells have a wider target scope. The data suggest that the efficacy of gene editing in immune cells was 15–45%, and the modified cells could survive long in the peripheral blood and tumor tissues of patients. After three or four months, some T-cells became central T-cells. These encouraging findings pave the way for future experimental cancer research utilizing CRISPR technology.

Farnesol restores wild-type colony morphology to 96% of Candida albicans colony morphology variants recovered following treatment with mutagens

Genome ◽  
2006 ◽  
Vol 49 (4) ◽  
pp. 346-353 ◽  
Author(s):  
Ellen C Jensen ◽  
Jacob M Hornby ◽  
Nicole E Pagliaccetti ◽  
Chuleeon M Wolter ◽  
Kenneth W Nickerson ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Quorum Sensing ◽  
Environmental Conditions ◽  
Budding Yeast ◽  
Colony Morphology ◽  
Morphological Transition ◽  
Strongly Correlated ◽  
Wild Type ◽  
Hyphal Morphology

Candida albicans is a diploid fungus that undergoes a morphological transition between budding yeast, hyphal, and pseudohyphal forms. The morphological transition is strongly correlated with virulence and is regulated in part by quorum sensing. Candida albicans produces and secretes farnesol that regulates the yeast to mycelia morphological transition. Mutants that fail to synthesize or respond to farnesol could be locked in the filamentous mode. To test this hypothesis, a collection of C. albicans mutants were isolated that have altered colony morphologies indicative of the presence of hyphal cells under environmental conditions where C. albicans normally grows only as yeasts. All mutants were characterized for their ability to respond to farnesol. Of these, 95.9% fully or partially reverted to wild-type morphology on yeast malt (YM) agar plates supplemented with farnesol. All mutants that respond to farnesol regained their hyphal morphology when restreaked on YM plates without farnesol. The observation that farnesol remedial mutants are so common (95.9%) relative to mutants that fail to respond to farnesol (4.1%) suggests that farnesol activates and (or) induces a pathway that can override many of the morphogenesis defects in these mutants. Additionally, 9 mutants chosen at random were screened for farnesol production. Two mutants failed to produce detectable levels of farnesol.Key words: farnesol-remedial mutants, farnesol-sensing mutants, farnesol-synthesis mutants, quorum sensing, Candida albicans, morphological transition.

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