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Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010s, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has rapidly been developed into a robust, multifunctional genome editing tool with many uses. Following the discovery of the initial CRISPR/Cas-based system, the technology has been advanced to facilitate a multitude of different functions. These include development as a base editor, prime editor, epigenetic editor, and CRISPR interference (CRISPRi) and CRISPR activator (CRISPRa) gene regulators. It can also be used for chromatin and RNA targeting and imaging. Its applications have proved revolutionary across numerous biological fields, especially in biomedical and agricultural improvement. As a diagnostic tool, CRISPR has been developed to aid the detection and screening of both human and plant diseases, and has even been applied during the current coronavirus disease 2019 (COVID-19) pandemic. CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases, including cancers, and has aided drug development. In terms of agricultural breeding, precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins, starch, oil, and other functional components for crop improvement. Adding to this, CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators. Looking to the future, increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology. This review provides an in-depth overview of current CRISPR development, including the advantages and disadvantages of the technology, recent applications, and future considerations.
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Humans , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Crops, Agricultural/genetics , Gene Editing/methods , Genetic Therapy , Nobel Prize , Plant BreedingABSTRACT
Objective To verify whether β-arrestin-2 inhibits autophagy by up-regulating PI3K/Akt signal to protect the liver from ischemia-reperfusion injury (IRI) in mice. Methods Twelve β-arrestin-2 knockout (KO) and twelve wild-type (WT) C57BL/6 mice were randomly divided into the KO+sham group, KO+IRI group, WT+sham group and WT+IRI group, six mice in each group. The mouse models with 70% liver IRI were established or sham operation was performed. Relevant experiments were carried out at 6 h after liver reperfusion or operation. The expression levels of apoptosis signal protein cleaved Caspase-3, proliferation signal protein Ki-67 and the PI3K/Akt signal protein p-Akt were detected by immunohistochemical staining. Results Immunohistochemical staining demonstrated that compared with the corresponding sham group, the positive cell count for cleaved Caspase-3, Ki-67 and p-Akt in liver tissues of mice was significantly increased in the KO+IRI and WT+IRI groups (all P < 0.01). Compared with the WT+IRI group, the positive cell count for cleaved Caspase-3 in liver tissues of mice was significantly increased, whereas the positive cell count forKi-67 and p-Akt was significantly decreased in the KO+IRI group (both P < 0.05). Conclusions β-arrestin-2 can mitigate the liver cell apoptosis and promote the repair of injury after IRI in mice. Moreover, β-arrestin-2 inhibits autophagy by up-regulating the PI3K/Akt signal to alleviate liver IRI in mice.
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Objective To analyze tumor immune microenvironment and related mechanisms in liver cancer.Methods We included 10 cases of hepatocellular carcinoma,hepatitis B patients and healthy volunteers from January 2015 to December 2017 in Shanxi Grand Hospital.We first detected the peripheral and local GM-CSF level in each group,detected myeloid-derived suppressor cells (MDSCs) GM-CSF and pathway-related protein expression.from liver cancer,tumor margin and normal liver tissue through flow cytometry and immunohistochemistry,Finally,we transfected the CCR4-NOT transcriptional complex subunit 7 (CNOT7) recombinant plasmid in the hepatoma cell line,and then detected the related protein expression.Results There was no significant difference for peripheral blood GM-CSF level between liver cancer group,hepatitis group and control group (P＞0.05).The level of local GM-CSF was (32.2±8.9) ng/L,which was higher than that of hepatocellular carcinoma (9.7±2.7) ng/L and normal liver tissue (11.6±2.9) ng/L.The difference was statistically significant (P＜0.05).The proportion of MDSCs at the edge of the tumor was (9.9 ±3.6) ％,which was higher than that of liver cancer (4.0± 1.5) ％ and normal liver tissue (6.3±2.3) ％,and the difference was statistically significant (P＜0.05).Immunohistochemistrydata was consistent with previous data.Compared with normal liver tissue,CNOT7 and STAT3 were highly expressed in liver cancer tissues,while STAT1 was lowly expressed.HepG2 human hepatoma cells were selected for transfection.Compared with the empty plasmid group,CNOT7 expression was decreased in the knocking out group at the same time STAT1 expression was increased,STAT3 and GM-CSF expression was decreased.Conclusion In hepatocellular carcinoma,the secretion of GM-CSF increased and the number of MDSCs increased.Knocking out CNOT7 reduced GM-CSF secretion and activate the JAK/STAT signaling pathway.
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Few tools of gene editing have been developed in Bacillus licheniformis at present. In order to enrich the tools, an FLP/FRT gene editing system that can repeatedly use a single selectable marker was constructed in Bacillus licheniformis, and the system was verified by knocking out an alpha amylase gene (amyL), an protease gene (aprE) and knocking in an exogenous Vitreoscilla hemoglobin gene (vgb). First, knock-out plasmids pNZTT-AFKF of amyL and pNZTT-EFKF of aprE were constructed using thermosensitive plasmid pNZT1 as a carrier. The two knock-out plasmids contained respective homology arms, resistance genes and FRT sites. Then the knock-out plasmids were transformed into Bacillus licheniformis and the target genes were replaced by respective deletion cassette via twice homologous exchange. Finally, an expression plasmid containing FLP recombinase reading frane was introduced and mediated the excision of resistance marker. In order to expand the practicability of the system, knock-in plasmid pNZTK-PFTF-vgb was constructed, with which knock-in of vgb at pflB site was carried out successfully. The results showed that amyL and aprE were successfully knocked out and the marker kanamycin cassette exactly excised. The activities of amylase and protease of deletion mutants were reduced by 95.3% and 80.4% respectively. vgb was successfully knocked in at pflB site and the marker tetracycline cassette excised. The expression of integrated vgb was verified via real-time PCR. It is the first time to construct an FLP/FRT system for gene editing in Bacillus licheniformis, which could provide an effective technical means for genetic modification.
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Bacillus licheniformis , Gene Editing , Plasmids , Sequence DeletionABSTRACT
Background: Zymomonas mobilis is a Gram-negative microaerophilic bacterium with excellent ethanol-producing capabilities. The RecET recombination system provides an efficient tool for direct targeting of genes in the bacterial chromosome by PCR fragments. Results: The plasmids pSUZM2a-RecET and pSUZM2a-RecE588T were first developed to co-express RecE or RecE588 and RecT for homologous recombination. Thereafter, the PCR fragments of the tetracycline resistance marker gene flanked by 60 bp of adhA (alcohol dehydrogenase I) or adhB (alcohol dehydrogenase II) homologous sequences were electroporated directly into ZM4 cells harboring pSUZM2a-RecET or pSUZM2a-RecE588T. Both adhA and adhB were replaced by the tetracycline resistance gene in ZM4, yielding two mutant strains, Z. mobilis ZM4 ΔadhA and Z. mobilis ZM4 ΔadhB. These two mutants showed varying extent of reduction in ethanol production, biomass generation, and glucose metabolism. Furthermore, enzyme activity of alcohol dehydrogenase II in Z. mobilis ZM4 ΔadhB exhibited a significant reduction compared to that of wild-type ZM4. Conclusion: This approach provided a simple and useful method for introducing mutations and heterologous genes in the Z. mobilis genome.
Subject(s)
Zymomonas/genetics , Homologous Recombination , Plasmids , Recombination, Genetic , Alcohol Dehydrogenase/metabolism , Zymomonas/enzymology , Electroporation , Ethanol/metabolism , Gene Knockout Techniques , MutationABSTRACT
Objective To evaluate the different change in atherosclerosis between apolipoprotein E knockout ( ApoE-/-) mice and low density lipoprotein receptor knockout ( LDL-R-/-) mice in different diet by vivo ultrasound biomicroscopy(UBM) . Methods Thirty-two 8 weeks age ApoE-/- mice and twenty-eight 20 weeks age LDL-R-/- mice were selected ,and each mouse model were divided into four groups randomly according to weight . Two groups of each model were fed with normal diet for 8 weeks and 16 weeks respectively as general groups ,and two other groups were fed with west diet as high-fat groups . Intima-media thickness ( IM T ) in the aortic root of all groups were assessed by Vevo 770 UBM , and all measurementsofUBMwerecomparedwithhistopathologyandblood-fat.Results ①Thickenwallor plaque in aortic root showed by UBM and histopathology could be found in ApoE-/- mice in all diet conditions ,but the changes in LDL-R-/- mice only could be found in high-fat diet conditions;② IM T of ApoE-/- mice measured in histopathology were significantly higher than LDL-R-/- mice in all diet condition ( P<0 .05) ,but in UBM ,the IMT of ApoE-/- mice showed significantly higher than that of LDL-R-/- mice only in general diet groups and 8 weeks high-fat diet group ( P <0 .05) ;③In blood-fat ,the total cholesterol ( TC) in ApoE-/- mice were significantly higher than that of LDL-R-/- mice in all diet conditions ( P <0 .05) . But triglyceride ( TG) was higher only in general diet when it was compared between ApoE-/- mice and LDL-R-/- mice . Conclusions It is recommend to select ApoE-/- mice as animal model in atherosclerosis study rather than LDL-R-/- mice ,because the higher serum TC and higher susceptible of atherosclerosis in ApoE-/- mice . The west diet is necessary if LDL-R-/- mice was used to atherosclerosis study .
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Objective To investigate the role of Glial cells missing 2 (Gcm2) in pathogenesis of hypoparathyroidism by knocking out Gcm2 gene in adult mice.Methods Tamoxifen was used to induce conditional knock-out of Gcm2 gene in Gcm2E2fl/flCre-ER mice.Genotypes of knock-out mice were identified by PCR.The protein expression level of Gcm2 was measured by Western blotting.The serum calcium and phosphorus were detected by the calcium and phosphorus assay kits, and the serum parathyroid hormone (PTH) level was detected by ELISA.Parathyroid cell proliferation was tested by Ki-67 immunohistochemical assay.The mRNA expression levels of PTH and calcium sensing receptor (CaSR) were detected by Real-time PCR.Bone mineral density was detected by micro CT.Results Gcm2 gene of parathyroid was confirmed to be knocked out by PCR.Compared with wild type and solvent control groups, Gcm2 knock-out group showed markedly lower protein expression of Gcm2, notably higher serum phosphorus and lower serum calcium and PTH concentrations (all P<0.01).The proliferation of parathyroid cells in Gcm2 knock-out mice were significantly higher(both P<0.01).The mRNA levels of PTH and CaSR in parathyroid gland of the knock-out group were significantly reduced (all P<0.01).Bone mineral density was significantly higher in Gcm2 knock-out group (all P<0.01).Conclusion Knockout of Gcm2 can lead to hypoparathyroidism in adult mice, indicating that Gcm2 is probably a therapeutic target for hypoparathyroidism.
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Objective To investigate the role of non-small cell lung cancer metastasis-related Trim72 gene in hepatocellular carcinoma (HCC) using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 system.Methods Hepa1-6 (Cas9) HCC cells were established with stable expression of Cas9 protein,and then specific gene knockout was performed using sgRNA targeting Trim72 gene.After obtaining the Hepa1-6 (Trim72-KO) cells,the metastasis and invasion abilities of the cells were evaluated by in vitro Transwell assay and in vivo subcutaneous lung metastasis examination.Results Hepa1-6 (Trim72-KO) cell line was successfully established by the CRISPR-Cas9 system.Transwell assay indicated that the mobility of Hepa1-6 (Trim72-KO) cells was increased compared to the control cells.Transwell assay indicated that the metastasis and invasion of Hepa1-6 (Cas9) HCC cells were enhanced after the knockout of Trim72 gene.The pathological examination of lung metastasis of subcutaneous tumor in vivo showed that the subcutaneously metastatic ability of Hepa1-6 (Trim72-KO) cells (the experimental group) was significantly stronger than Hepa1-6 (Cas9) cells (the control troup) that were not transferred to the corresponding sgRNA.Conclusions The trim72 gene knocked-out HCC cells were obtained by CRISPR-Cas9 system,which showed stronger metastasis and invasion abilities than the control cells.It is suggested that Trim72 gene may play an important role in the invasion and metastasis of HCC,and Trim 72 gene is expected to be a potential target for gene therapy of liver cancer.
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Objective To observe the protective effect and mechanism of 2,3,5,4 '-tetrahydroxystilbene-2-O-beta-D-glucoside ( THSG) on atherosclerosis in ApoE konck-out mice. Methods A total of 24 ApoE knock-out mice were randomly divided into normal control group (n=8), model control group (HFD, high-fat diet, n=8) and treated group (THSG, 20 mg· kg-1, i. g. , n=8). The atherosclerosic plaque of aorta wall and aorta root were measured by oil red O staining;The expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in human umbilical vein endothelial cells (HUVEC) through C-reaction protein ( CRP) was studied by Western blotting. Results The atherosclerosis plaque in normal control group was not observed. The lipid accumulation decreased in the aorta and the plaque areas in the aortic sinus in THSG treated-group compared with model control group. Moreover, THSG down-regulated CRP-induced LOX-1 expression in HUVEC. Conclusion The atheroscletosis plaque in ApoE knock-out mice was decreased by THSG. The mechanism might be related to the inhibition of the expression of LOX-1 protein.
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Objective To explore the role of 18 ku translocator protein (TSPO) in the anti-post-traumatic-stress-disorder(PTSD) effects of YL-IPA08 and the value of TSPO as a potential pharmacological target using gene knock out mice.Methods The PCR method was used to genotype TSPO wild type (WT) mice and knock out (KO) mice.Foot shock was used to establish a well-accepted mouse model of PTSD,the open field test (OFT) was used to evaluate the locomotor activity in mice,and freezing measurement was used to evaluate the PTSD-like fear behavior in mice.Results Compared with TSPO WT mice,KO mice had no expressible TSPO gene,but showed similar locomotor activity to WT mice after PTSD modeling.On day 1,day 5 and day 16 after PTSD modeling (day-1-day 0),both WT and KO mice showed significant PTSD-like behavior with enhanced freezing time.However,8 d treatment (day 0-day 7) of YL-IPA08 (0.3 mg/kg,once daily) or positive drug sertraline (15 mg/kg,once daily) after PTSD modeling significantly reduced freezing time selectively in WT mice,but not in KO mice.Conclusion It has been found for the first time that TSPO WT and KO mice can show the same sensitivity to PTSD modeling (namely the same PTSD-like behavior performance).Interestingly,TSPO can mediate the anti-PTSD effects of YL-IPA08.Therefore,the present study provides direct evidence for the value of TSPO as an potential pharmacological target for PTSD.
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Objective CRISPR/Cas9 genome-editing technique provides an novel method for whole genome editing in eukaryotic cells.Recently,we found that gene subtype library with smaller size and focused pur-pose is more economical and practical. In this study,we aimed to target kinases,a group of pivotal cell signal transducers,to construct a kinase knock-out library using CRISPR/Cas9 technique.The construction strategy wll al-so be discussed. Methods 10 sgRNA was designed for each kinase target.After oligo pool synthesis by semicon-ductor chip,the oligos were eluted from the chip. The oligo templates were amplified and cloned into Cas9 vector and transformed into Stble3 competent cells.Monoclonal colonies were selected for DNA sequencing. Results(1) GO analysis of 507 cell kinases showed that the cell kinases took part in a wide range of cell signaling.(2)The sgRNA pool with about 140 bp in length was successfully amplified by using oligo pool as the template and univer-sal PCR primers.(3)In 40 identified library clones,34 clones were sequenced successfully. Among them,the DNA sequencing results of 25 samples were completely consistent with the designed target sequences.But there are some mutations in the primers of 9 samples.Failure in bacteria shaking,DNA sequencing and other factors were ex-isted in the other clones. Conclusion The CRISPR/Cas9 kinase knock-out library can be widely used for screen-ing the important kinases which may mediate cell proliferation,metastasis,drug resistance and autophagy.This li-brary will play an important role in clarifying the development of disease associated with kinases.
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Objective · To evaluate the influence of cathepsin S(CatS) on the severity of communicating hydrocephalus in a kaolin injected mouse model.Methods · Kaolin suspension was injected to 8 CatS knock-out (CatS -/-) mice and 12 wild type (WT) C57BL/6 mice through cisterna magna to establish communicating hydrocephalus mouse model. Cerebral magnetic resonance imaging (MRI) was used before and 1 week after kaolin injection to compare lateral ventricular volume. Lateral ventricular index was calculated to analyze the severity of hydrocephalus. Results · One week after kaolin injection,1 in CatS -/- group and 2 in WT group died. The mortality rate was 12.5% each and there was no significant difference (P=1.000). MRI results showed varying degrees of ventriculomegaly in both groups. Lateral ventricular index of CatS -/-group (n=8) and WT group (n=16) before kaolin injection was 0.05±0.01 and 0.04±0.01 respectively (P=0.720). One week after kaolin injection, lateral ventricular index of CatS-/- group (n=7) and WT group (n=14)was 0.13±0.02 and 0.11±0.01 respectively (P=0.950). In each group, in 71.4% of mice, lateral ventricular index enlarged twice or more. Conclusion · One week after kaolin injection into cisterna magna, lateral ventricles enlarges obviously, indicating hydrocephalus occurs, with high success rate. CatS gene deficiency has no significant influence on the development of communicating hydrocephalus.
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Objective · To evaluate the influence of cathepsin S(CatS) on the severity of communicating hydrocephalus in a kaolin injected mouse model.Methods · Kaolin suspension was injected to 8 CatS knock-out (CatS -/-) mice and 12 wild type (WT) C57BL/6 mice through cisterna magna to establish communicating hydrocephalus mouse model. Cerebral magnetic resonance imaging (MRI) was used before and 1 week after kaolin injection to compare lateral ventricular volume. Lateral ventricular index was calculated to analyze the severity of hydrocephalus. Results · One week after kaolin injection,1 in CatS -/- group and 2 in WT group died. The mortality rate was 12.5% each and there was no significant difference (P=1.000). MRI results showed varying degrees of ventriculomegaly in both groups. Lateral ventricular index of CatS -/-group (n=8) and WT group (n=16) before kaolin injection was 0.05±0.01 and 0.04±0.01 respectively (P=0.720). One week after kaolin injection, lateral ventricular index of CatS-/- group (n=7) and WT group (n=14)was 0.13±0.02 and 0.11±0.01 respectively (P=0.950). In each group, in 71.4% of mice, lateral ventricular index enlarged twice or more. Conclusion · One week after kaolin injection into cisterna magna, lateral ventricles enlarges obviously, indicating hydrocephalus occurs, with high success rate. CatS gene deficiency has no significant influence on the development of communicating hydrocephalus.
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Objective:To build the model of the gene FKBP38 (FK506 binding protein 38) conditional knock out in liver.Methods:Transgenic mouse whose FKBP38 gene was flanked with loxP was constructed by embryo microinjection.The FKBP38 gene was deleted by breeding mice harboring two loxP sites in FKBP38 (FKBP38fl/fl) with the mice bearing the expression ofCre recombinase mice driven by an album promoter.Afterward,the genotype of FKBP38 conditional knockout mice was analyzed.Results:①Relative hepatic FKBP38 mRNA levels showed significant difference between FKBP38 conditional knockout mice (FKBP38-/-) and wild type(P＜ 0.001).②Relative hepatic FKBP38 protein expression levels of FKBP38 conditional knockout mice (FKBP38-/-) were significantly different with wild type(P＜0.001).③Relative phosphorylation of hepatic p70 S6K and 4E-BP-1 protein of FKBP38 conditional knockout mice (FKBP38-/-) showed no significant difference,with slight decrease in phosphorylation of 4E-BP-1,compared with wild type.④No significant difference in expression of hepatic Bcl-2 between FKBP38-/-and wild type.Conclusions:The mouse model of the gene FKBP38 (FK506 binding protein 38) conditional knock out in liver is successfully built.
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To study the biological function of DNAH2 (Homo sapiens dynein, axonemal, heavy chain 2) gene, we constructed human stable U2OS cell line of DNAH2 gene knockout through CRISPR/Cas9n double nick system. The A, B sgRNAs (Single guide RNA) and complementary strands were designed and synthesized. The double-stranded structures were formed during annealing, and connected with BbsⅠ cohesive ends-containing pX462 linear vector to construct the recombinant eukaryotic expression plasmids, including pX462-DNAH2-A and pX462-DNAH2-B. After the co-transfection of the two plasmids into U2OS cells, the addition of puromycin and limiting dilution method were used to obtain positive monoclonal cell line. Western blotting assay was then performed to detect the expression of DNAH2 protein, and PCR-sequencing technology was finally utilized to analyze the mutation feature. The results showed that A, B sgRNAs duplex was successfully inserted into pX462 vector, and DNAH2 protein was not expressed and DNAH2 gene suffered from the frame-shift mutation in U2OS-DNAH2-KO monoclonal cell line. These demonstrated that DNAH2 knockout U2OS stable cell line was successfully constructed through CRISPR/Cas9n double nick system, which providing a useful tool for the study of DNAH2 gene.
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Objective To preliminarily explore the effects of gene knock-out and overexpression of aldolase A on tumor cell proliferation,and then confirm the relationship between aldolase A and tumor growth during the process of anaerobic metabolism. Methods Through vector plasmid building and lentiviral infection,the cell lines stably overexpressing aldolase A and its gene knock-out cell lines were constructed respectively. The restructured cell lines were validated on gene and protein levels. Then ,the changes of cell proliferation ratio were observed. Results Real time PCR results showed that aldolase A gene expression of the overex?pressing cell line was about 3 times that of the normal cells. Among the five interfering targets of aldolase A designed according to aldol?ase A RNA sequences,aldolase A4 had more obvious interference effect on the target gene expression,the aldolase A mRNA level of which was about 1/50 of the normal cells. Compared with normal control group,the proliferation rate of aldolase A gene overexpression significantly increased by 40% in 48 h(P<0.05),while the proliferation rate of aldolase A gene knock out cells significantly de?creased about 86%in 24 h(P<0.05). Conclusion Aldolase A expressions have obvious effects on hepatocellular carcinoma cells.
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Objective: To prepare the glycyrrhizic acid (GA) immunoaffinity chromatography column, which could specifically knock out the GA. Methods: An immunoaffinity chromatography (IAC) column was developed by covalently coupling the anti-GA-MAb to CNBr-activated Sepharose™ 4B. The concentration of GA was detected by HPLC, and the maximum coupling capacity, stability, precision, and accuracy of the IAC column for GA were studied. Results: The maximum capacity of the IAC column for GA was 1.326 11 mg, the precision (RSD) of GA was 0.65%, and the accuracy (RE) was 0.37%. RSD of GA stayed 1.37% during 32 d, which showed a pretty well stability. Conclusion: GA-IAC column could rapidly, effectively, and stably knock out the GA.
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Objective To preliminarily explore the effects of gene knock-out and overexpression of aldolase A on tumor cell proliferation, and then confirm the relationship between aldolase A and tumor growth during the process of anaerobic metabolism. Methods Through vector plasmid building and lentiviral infection, the cell lines stably overexpressing aldolase A and its gene knock- out cell lines were constructed respectively. The restructured cell lines were validated on gene and protein levels. Then, the changes of cell proliferation ratio were observed. Results Real time PCR results showed that aldolase A gene expression of the overexpressing cell line was about 3 times that of the normal cells. Among the five interfering targets of aldolase A designed according to aldolase A RNA sequences, aldolase A4 had more obvious interference effect on the target gene expression, the aldolase A mRNA level of which was about 1/50 of the normal cells. Compared with normal control group, the proliferation rate of aldolase A gene overexpression significantly increased by 40% in 48 h(P<0.05), while the proliferation rate of aldolase A gene knock out cells significantly de? creased about 86% in 24 h(P<0.05). Conclusion Aldolase A expressions have obvious effects on hepatocellular carcinoma cells.
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Periaxin, a protein of noncompact myelin, is specifically expressed in the peripheral nervous system (PNS). There are two protein isoform L-periaxin and S-Periaxin by alternative splicing of periaxin gene, playing an important role in the initiation of myelin formation. So far, 18 different mutation sites in L-periaxin gene have been found to induce the peripheral demyelinating neurological charcot-marie-tooth diseases subtype 4F (CMT4F). The technique of activation of transcription activator-like effector nucleases (TALENS) was used to knock out the L-periaxin gene in RSC 96 cell line of Rattus. According to the design principle, the knock-out site of L-periaxin was assured to NLS domain of L-periaxin, which is target sequence of left and right arms of TALEN. The knock-out vectors of TALEN-L and TALEN-R were established and transfected into RSC96 cell. After puromycin screening, L-periaxin was knocked out successfully in RSC96 cell, which is confirmed by DNA sequence. The mutation efficiency is 21.6%. S-periaxin, not L-periaxin can be detected by Western blotting in L-periaxin gene knock-out RSC96 cell. The cell growth rate was decreased and the number of cells in G1 increased and decreased in S phase in L-periaxin gene knock-out RSC96 cell by flow cytometry and MTT assay.
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Animals , Rats , Cell Line , Charcot-Marie-Tooth Disease , Genetics , Gene Knockout Techniques , Membrane Proteins , Genetics , Mutation , Protein IsoformsABSTRACT
This study investigated the effects of SIRT1 gene knock-out on osteoarthritis in mice, and the possible roles of SREBP2 protein and the PI3K/AKT signaling pathway in the effects. Mice were randomly divided into a normal group and a SIRT1 gene knock-out group (6 mice in each group). In these groups, one side of the knee anterior cruciate ligament was traversed, and the ipsilateral medial meniscus was cut to establish an osteoarthritis model of knee joint. The countralateral synovial bursa was cut out, serving as controls. The knee joint specimens were then divided into four groups: SIRT1control group (group A, n=6); SIRT1osteoarthritis group (group B, n=6); SIRT1control group (group C, n=6); SIRT1osteoarthritis group (group D, n=6). HE staining, Masson staining, Safranin O-Fast Green staining and Van Gieson staining were used to observe the morphological changes in the articular cartilage of the knee. Immunohistochemical staining was employed to detect the expression of SIRT1, SREBP2, VEGF, AKT, HMGCR and type II collagen proteins. SA-β-gal staining was utilized to evaluate chondrocyte aging. The results showed clear knee joint cartilage destruction and degeneration in the SIRT1osteoarthritis group. The tidal line was twisted and displaced anteriorly. Type II collagen was destroyed and distributed unevenly. Compared with the SIRT1osteoarthritis group and SIRT1control group, SIRT1 protein expression was not obviously changed in the SIRT1osteoarthritis group (P>0.05), while the expression levels of the SREBP2, VEGF and HMGCR proteins were significantly increased (P<0.05) and the levels of AKT and type II collagen proteins were significantly decreased (P<0.05). SIRT1 gene knock-out may aggravate cartilage degeneration in osteoarthritis by activating the SREBP2 protein-mediated PI3K/AKT signalling pathway, suggesting that SIRT1 gene may play a protective role against osteoarthritis.