Establishment of lens opacity model in Grx2 knockout mice based on CRISPR/cas9 system and the role of Grx2 in the pathogenesis of cataract / 中华实验眼科杂志

Objective:To explore the role of Grx2 in the pathogenesis of cataract by establishing Grx2 knockout (KO) and knockin (KI) mouse models. Methods:Ten black C57BL/6J mice were selected to make Grx2 KO model ( n=5) and Grx2 KI model ( n=5) using CRISPR/Cas9 system.The offspring mice were sequenced by tail clipping and included in the corresponding experimental group according to the genotype.The general condition and lens opacity was recorded.After the mice were sacrificed, the pathological changes of lens were observed by hematoxylin-eosin staining.The contents of reactive oxygen species (ROS) and 8-hydroxy-desoxyguanosine (8-OHdG) were analyzed by enzyme-linked immunosorbent assay (ELISA).The relative expression levels of Grx2, glutathione (GSH), B-cell lymphoma-2 (Bcl-2) , glutathione disulfide (GSSG) and Bcl-2-associated X protein (Bax) in mice lens were assayed.The use and feeding of experimental animals were in accordance with the Regulations on the Management of Experimental Animals issued by the State Science and Technology Commission.The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University (No.2020-125). Results:The offspring of Grx2 KO and Grx2 KI homozygous and heterozygous mice were confirmed by tail cutting nested PCR and gene sequencing.Compared with the wild type (WT) mice of same age, the lens opacity of Grx2 KO heterozygous mice occurred earlier, while the lens of Grx2 KI homozygous mice remained transparent all the time.A large number of gaps and vacuoles were found in the lens fibers of 5-month-old Grx2 KO mice.The 8-OHdG content and ROS fluorescence intensity in the lens of 5-month-old Grx2 KO mice were (3.886±0.326)ng/ml and 1 594±132, which were significantly higher than (3.531±0.250)ng/ml and 1 157±123 in WT mice ( t=2.711, P=0.033; t=3.384, P=0.028).The relative expression levels of Grx2, GSH and Bcl-2 in the lens of 5-month-old Grx2 KO mice were 0.23±0.01, 0.70±0.06 and 0.32±0.03, which were significantly lower than 0.52±0.02, 1.04±0.08 and 0.49±0.04 of WT mice ( t=2.815, P=0.020; t=2.457, P=0.033; t=2.279, P=0.041). Conclusions:Grx2 KO and Grx2 KI mouse models are successfully established in this study.The occurrence and development of age-related cataract are accelerated in Grx2 KO mice.

Application of CRISPR/Cas9 lentiviral vector in construction of rat hepatic stellate cells with COX-2 gene knockout / 临床肝胆病杂志

ObjectiveTo obtain HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/- cells with COX-2 gene defect by transfecting HSC-T6 cells with CRISPR/Cas9 lentiviral vector, and to provide a good method for further functional research and new strategies for the clinical treatment of liver fibrosis. MethodsThe COX-2 gene-specific sgRNAs (COX-2-sgRNA-1, COX-2-sgRNA-2, COX-2-sgRNA-3) were designed, synthesized, and connected to the GV371 vector, and the recombinant plasmid and the packaging plasmid were transfected into 293T cells to form lentivirus particles; the fluorescence method was used to measure virus titer. The most appropriate amount of the virus was calculated based on MOI. Lenti-Cas9-puro was transfected into HSC-T6 cells, and HSC-T6-Cas9 cells were screened out by puromycin; Lenti-COX-2-sgRNA-EGFP was transfected into HSC-T6-Cas9 cells to obtain HSC-T6-COX-2-/- cells. Cruiser enzyme digestion and Western blot were used to verify gene knockout at the gene and protein levels. An analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsSequencing verified that the COX-2-sgRNA expression vector was constructed successfully. Recombinant expression plasmids and packaging plasmids were transfected into 293T cells to form lentivirus particles, and the fluorescence method showed a virus titer of ＞1×108. HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/- cells with COX-2 gene defect were successfully constructed. The HSC-T6-Cas9 group had significantly higher relative mRNA expression of LV-Cas9-Puro than the CON group (541.93±105.76 vs 1.00±0.02, t=12.995, P＜0.01). Cruiser enzyme digestion and Western blot showed that the CRISPR/Cas9 lentivirus expression vectors played a role in the target, among which COX-2-sgRNA-2 knockout had the most significant effect, and this group had a significant reduction in the protein expression level of COX-2 compared with the CON group and the NC group (both P＜0.05), suggesting that COX-2-sgRNA was active. ConclusionA CRISPR/Cas9 lentivirus vector is successfully constructed for COX-2 target gene, and HSC-T6-COX-2-/- cells with stable COX-2 gene knockout are obtained.

Influence of abaR gene knockout on growth metabolism and biofilm formation of Acinetobacter baumannii / 中华烧伤杂志

Objective@#To investigate the influence of abaR gene knockout on growth metabolism and biofilm formation of Acinetobacter baumannii.@*Methods@#The abaR gene was knocked out from Acinetobacter baumannii standard strain ATCC 17978 (wild strain) by homologous recombination method, and then the ATCC 17978 abaR knockout strain (ATCC 17978/ΔabaR: : Kn) was obtained and verified by polymerase chain reaction (PCR) electrophoresis and sequencing. The growth curves of Acinetobacter baumannii wild strain and Acinetobacter baumannii knockout strain were determined by microplate reader within cultivation hour (CH) 18, and the biofilm formation ability was measured by crystal violet staining at CH 8, 24, and 48, respectively. The sample number at each time point was 3.The results were denoted as absorbance value. Data were processed with analysis of variance of factorial design, one-way analysis of variance, t test, and least-significant difference test.@*Results@#(1) The length of PCR product of target fragment ΔabaR: : Kn was 3 029 bp. The abaR gene was knocked out to obtain the knockout strain ATCC 17978/ΔabaR: : Kn. The length of PCR product of the knockout strain was 3 300 bp. The abaR gene was successfully knocked out. (2) At CH 2, 3, and 4, the absorbance values of Acinetobacter baumannii wild strain were slightly higher than those of the knockout strain. The absorbance values of Acinetobacter baumannii wild strain and knockout strain were similar from CH 5 to 18. (3) At CH 8 and 24, the biofilm formation ability of Acinetobacter baumannii wild strains (0.644±0.066, 0.574±0.184) was similar to that of knockout strains (0.559±0.008, 0.394±0.030, t=2.209, 1.167, P>0.05). At CH 48, the biofilm formation ability of Acinetobacter baumannii wild strains (1.157±0.259) was significantly stronger than that of Acinetobacter baumannii knockout strains (0.576±0.026, t=3.865, P<0.05). The biofilm formation ability of Acinetobacter baumannii wild strains at CH 48 was significantly stronger than that at CH 8 and 24 (P<0.05). The biofilm formation ability of Acinetobacter baumannii knockout strains at CH 24 was significantly weaker than that at CH 8 and 48 (P<0.05).@*Conclusions@#The abaR gene of Acinetobacter baumannii ATCC 17978 can be successfully knocked out by homologous recombination to obtain its knockout strain ATCC 17978/ΔabaR: : Kn. The abaR gene does not affect the growth and metabolism of Acinetobacter baumanniibut can weaken its biofilm formation ability.

Resistance of osteosclerotin knockout mice to glucocorticoid induced bone microstructure degeneration / 中国医师杂志

Objective@#To observe the changes in bone mineral density and microstructure parameters in sclerostin (SOST) gene knockout mice treated with glucocorticoid.@*Methods@#12 4-week-old SOST knockout mice were randomly divided into two groups (n=6): methylprednisolone intervention group [SOM group, methylprednisolone 3 mg/(kg·d), subcutaneous injection], placebo group (SOS group, isovolumetric saline subcutaneous injection). 12 wild-type mice were randomly divided into two groups (n=6): wild-type placebo group (WTS group, isovolumetric saline subcutaneous injection), wild methylprednisolone intervention group [WTM group, methylprednisolone 3 mg/(kg·d), subcutaneous injection]. 12 weeks later, mice were sacrificed and one lumbar vertebra of each mouse was selected for micro-CT analysis.@*Results@#There was no difference in bone mineral density (BMD), trabecular volume fraction, trabecular number and trabecular thickness between SOM and SOS groups (P>0.05). BMD, trabecular volume fraction, trabecular number and trabecular thickness in SOM and SOS groups were significantly higher than those in WTS and WTM groups (P<0.05). BMD, trabecular volume fraction, trabecular number and trabecular thickness in WTM group were significantly lower than those in WTS group (P<0.05).@*Conclusions@#Sclerotin gene knockout mice can resist glucocorticoid-induced bone loss and bone microarchitectural deterioeration. The treatment of osteoporosis with SOST/sclerotin as a target will be an effective method in the future.

Resistance of osteosclerotin knockout mice to glucocorticoid induced bone microstructure degeneration / 中国医师杂志

Objective To observe the changes in bone mineral density and microstructure parameters in sclerostin (SOST) gene knockout mice treated with glucocorticoid.Methods 12 4-week-old SOST knockout mice were randomly divided into two groups (n =6):methylprednisolone intervention group [SOM group,methylprednisolone 3 mg/(kg· d),subcutaneous injection],placebo group (SOS group,isovolumetric saline subcutaneous injection).12 wild-type mice were randomly divided into two groups (n =6):wild-type placebo group (WTS group,isovolumetric saline subcutaneous injection),wild methylprednisolone intervention group [WTM group,methylprednisolone 3 mg/(kg · d),subcutaneous injection].12 weeks later,mice were sacrificed and one lumbar vertebra of each mouse was selected for microCT analysis.Results There was no difference in bone mineral density (BMD),trabecular volume fraction,trabecular number and trabecular thickness between SOM and SOS groups (P ＞ 0.05).BMD,trabecular volume fraction,trabecular number and trabecular thickness in SOM and SOS groups were significantly higher than those in WTS and WTM groups (P ＜0.05).BMD,trabecular volume fraction,trabecular number and trabecular thickness in WTM group were significantly lower than those in WTS group (P ＜ 0.05).Conclusions Sclerotin gene knockout mice can resist glucocorticoid-induced bone loss and bone microarchitectural deterioeration.The treatment of osteoporosis with SOST/sclerotin as a target will be an effective method in the future.

The effect of AmpD on the expression of AmpC β-lactamase and the regulation mechanisms of β-N-acetylglucosaminidase in Yersinia enterocolitica / 中华预防医学杂志

Objective@#In this study, we analyze the regulation mechanisms of the expression of ampD in AmpC β-lactamase and the regulation mechanism of β-N-acetylglucosaminidase (NagZ) in Yersinia enterocolitica.@*Methods@#We construct the mutation strains of Yersinia enterocolitica AmpD (AmpD1-3) gene (ampD1-3), Low-Molecular-Mass Penicillin-Binding Proteins (LMM PBPs) gene (pbp4, pbp5a, pbp5b, pbp7), NagZ gene (nagZ), and ampR gene by deleting and complementing genes, and induce them by cefoxitin. We determined the activity of AmpC β-lactamase activity (U) of mutant strains (basal level and induce level) by using cephalothiophene hydrolysis method, the promoter activity of AmpC β-lactamase ((relative light unit (RLU)) was detected by the luxCDABEreporter system, and the activity of β-N-acetylglucosaminidase (nmol/L) was determined by by using 4-nitrophenyl N-acetyl-β-D-glucosaminide as the chromogenic substrate.@*Results@#AmpD1 (Basal level: (3.29±1.58) U; Induced level: (4.08±1.75) U) was the most potent one. The YEΔ5b, YEΔ4Δ5b, YEΔ5aΔ5b and YEΔ5bΔ7 of ampC promoter activity increase significantly, whichYEΔ4Δ5b is the highest one (Basal level: (106 903.16±61 910.61) RLU; Induced level: (205 427.45±45 352.17) RLU). The YEΔ4Δ5bΔ7 of ampC promoter activity is the highest among triple mutant strain (Basal level: (304 108.04±99 274.53) RLU; Induced level: (531 440.21±68 891.02) RLU). Quadruple deletion strain YEΔ4Δ5aΔ5bΔ7 have the highest ampC promoter activity (Basal level: (1 013 810.99±260 955.96) RLU; Induced level: (1 230 214.59±205 526.79) RLU). After the deletion of nagZ gene, there is no significant change in β-lactamase activity of YEΔD1D2D3ΔZ, while β-lactamase activity of YEΔ4Δ5aΔ5bΔ7ΔZ shows a significant decrease (Basal level: (0.30±0.20) U; Induced level: (0.29±0.21) U), which basically drops to the wild strain level.@*Conclusion@#This is the first report of ampC multi-step upregulation mechanism driven by three AmpD homologues in Yersinia enterocolitica. The AmpC regulation mode with the function of single PBP4, PBP5a or PBP7 is relatively low, which work in coordination with PBP5b. Yersinia enterocolitica have both NagZ-depend and NagZ-independent mechanisms for β-lactamase expression.

CNOT 7 gene induces HepG2 cellular immunologic tolerance against Vγ9Vδ2T cells / 中华普通外科杂志

Objective To study the action of CNOT 7 (CCR4-NOT transcription complex subunit 7 human)gene and its mechanisms in the process of Vγ 9Vδ2T cell immunologic tolerance of HepG2 cells (Hepatoblastoma G2 Cell Line).Methods The shCNOT 7 (Recombinant plasmid of CNOT 7) and control vector shRNA were transfected into HepG2 cells.Vγ9Vδ2T cytokine stimulated each group before and after cell transfection,Cell apoptosis was detected by flow cytometry (FCM),CNOT 7 protein and STAT1,STAT3 expression level was detected by Western blot.CNOT 7,STAT1 and STAT3 protein expression levels of HepG2 liver cancer cell lines and L02 normal liver cell line was assayed by Western blot.Results When stimulated by Vγ9Vδ2T cytokine,the apoptosis rate of gene-knockdown group significantly improved from (7.55％ ±2.63％) to (20.59％ ±3.12％).Compared with L02 cells,the CNOT7 protein expression of HepG2 cells increased (F =28.76,P ＜ 0.01),STAT3 protein expression increased (F =110.29,P ＜ 0.01),while STAT1 protein expression was down-regulated (F =35.67,P ＜ 0.01).CNOT 7 knockout could induce HepG2 cells STAT1 expression (t =6.69,P ＜ 0.05).Conclusions CNOT 7 gene could induce HepG2 cells Vγ 9Vδ2T cellular immune tolerance.CNOT 7 knockout could reverse the Vγ 9Vδ2T cell immunologic tolerance of HCC.

Cyclin-dependent kinase 7 gene silencing or knockout represses proliferation of ovarian cancer cells and the mechanism research / 肿瘤

Objective: To investigate the role of cyclin-dependent kinase 7 (CDK7) in the proliferation of ovarian cancer cells. Methods: The ovarian cancer cell lines OVCA433, TOV-112D and IGROVl were transfected with specific siRNA to downregulate the expression of CDK7 gene, then the cell proliferation viability was detected by CCK-8 method. The CDK7 gene in ovarian cancer HEY, OVCA420, OVCA433 and IGROVl cells was knocked out by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system, then the clone formation ability was detected by plate clone formation assay. The ovarian cancer TOV-112D, IGROV1, OVCA433, OVCAR8, OV90, SKOV3 and COV413B cells were treated with CDK7-inhibitor THZl, then the cell proliferation viability and clone formation ability were detected by CCK-8 method and clone formation assay, respectively. Furthermore, the expression level of CDK7 protein and the phosphorylation level of RNA polymerase n (RNAPolE) in IGROV1, OVCA433, SKOV3 and COV41 3B cells treated with THZl were analyzed by Western blotting. Results: The proliferation and clone formation abilities of various ovarian cancer cells were significantly decreased after CDK7 gene was silenced or knocked out (all P < 0.05). THZ1 repressed the proliferation and clon e formation of ovarian cancer cells, an d downregulated the expression of CDK7 and the phosphorylation of RNAPoln (all P < 0.05). Conclusion: CDK7 may promote the proliferation of ovarian cancer cells by regulating the phosphorylation of RNAPoln.

Quick establishmet of ApoE-/- rats atherosclerosis model by high-fat diet combined with balloon injury / 解放军医学杂志

Objective To quickly establish the To quickly establish the atherosclerosis (AS) model in apoprotein E gene knockout (ApoE-/-) rats by short-term high-fat diet combined with balloon injury. Methods Eight-week-old male SD and ApoE-/- rats (10 each) were used in the experiments. After fed with high-fat diet for 2 weeks, the blood routine, hepatorenal function, blood lipid profile and C-reactive protein (CRP) were measured, and then all the left common carotid arteries were processed by balloon injury. After 2 weeks, rats euthanasia were carried out by excessive chloral hydrate, and then the operation side common carotid arteries were stained with HE, Masson trichrome and oil red O. While the common carotid arteries accepted CD68, α-SMA immunofluorescent staining. Results The blood lipid level was significantly higher in ApoE-/- rats than in SD rats [total cholestrol (TC): 18.56±2.82mmol/L vs 5.69±1.98mmol/L, P<0.01; low density lipoprotein (LDL): 6.86±1.47mmol/L vs 1.92±0.76mmol/L, P<0.01]. In the condition of serious blood lipid disorders, the ApoE-/- rats had been in a state of inflammation [CRP: 4.66±0.57mg/L vs 0.39±0.21mg/L, P<0.05; white blood cell: (21.79±5.10)×109/L vs (14.82±2.41)×109/L, P<0.01; neutrophil: (9.28±3.35)×109/L vs (2.10±0.96)×109/L, P<0.01]. HE and Masson staining showed that obvious hyperplasia formed and collagen fibers deposited slightly in the two groups. Oil red O staining revealed the obvious hyperplastic plaques in ApoE-/- rats, but a negative result in SD rats. Immunofluorescence staining showed the significant positive CD68 and weak positive α-SMA in the plaque of ApoE-/- rats, while in SD rats the positive -SMA was pointed out with no CD68 coloration. Conclusion The atherosclerosis model of ApoE-/- rat may be quickly established by short-term high-fat diet combined with balloon injury.

Establishing mouse models of allergic rhinitis by knocking outH2-eb1 gene / 中国组织工程研究

BACKGROUND:HLA-DRB1 is related to the pathogenesis of alergic rhinitis. Construction ofHLA-DRB1 gene knockout animal models not only elucidates the pathogenesis of alergic rhinitis, but also provides a good way for the elucidation of the pathogenesis of alergic rhinitis-related diseases. OBJECTIVE:To establish the HLA-DRB1gene knockout animal models. METHODS:Homozygous, wild-type and heterozygous mice were obtained by inbreeding of the heterozygous mice. Confirmed by gene and protein identification, 24 female wild-type (H2-eb1+/+) mice and 12 H2-eb1-/-mice aged 8 weeks were selected according to the random number table. 12 H2-eb1+/+ mice and 12 H2-eb1-/- mice were sensitized with ovalbumin to establish the mouse models of alergic rhinitis. Another 12 mice were sensitized with PBS as comparison. RESULTS AND CONCLUSION:Compared with the control mice, serum levels of ovalbumin-specific IgE and interleukin-4 were significantly increased, while serum level ofγ-interferon was significantly decreased in the mouse models of alergic rhinitis. Serum levels of IgE and interleukin-4 were lower, while serumγ- interferon level was higher, inH2-eb1-/-gene knockout mice of alergic rhinitis than those in the H2-eb1+/+ gene knockout wild-type mice. These results suggest thatH2-eb1 gene may play an important role in regulating Th1/Th2 imbalance in the pathogenesis of alergic rhinitis.

Construction and application of Staphylococcus aureus gene knockout plasmid / 中国组织工程研究

BACKGROUND:Methicil in-resistant Staphylococcus aureus has been a primary pathogen of nosocomial infections worldwide. To construct a quick and easy knockout method is an important technique of studying virulence and resistance of methicil in-resistant Staphylococcus aureus. OBJECTIVE:To construct the Staphylococcus aureus gene knockout plasmid for understanding the antibiotic resistance and virulence of Staphylococcus aureus. METHODS:pUC19 was considered as a basic skeleton of construction. pLE194Ts temperature-sensitive replicon and tetracycline resistance gene fragment pHY300PLK plasmid in pCL52.1 were bound to EcoR I site in pUC19 by high assurance amplification. Al multiple clone sites in pUC19 were reserved. The Escherichia coli-Staphylococcus aureus shuttle plasmid was obtained. The N315 dapB gene knockout plasmid was obtained through gene knockout technology. This strain was eventual y identified by multiplex-PCR. RESULTS AND CONCLUSION:The Escherichia coli-Staphylococcus aureus shuttle plasmid, pYZ1 and pYZ8, was successful y constructed, and had been used in Staphylococcus aureus gene knockout. Homologous recombinant plasmid pYZ-ΔdapB was constructed by restriction enzyme digestion and overlap technique. After genetical y modification in RN4220, the constructed gene knockout plasmid pYZ-ΔdapB was introduced to N315 to be screened in the low culture temperature. The deletion strain was successful y obtained after being identified by multiplex-PCR. Above data suggested that pYZ1 and pYZ8 can be successful y used for Staphylococcus aureus gene detection, which provides a tool to study resistance and virulence of clinical Staphylococcus aureus strains.

Rational design of diagnostic and vaccination strategies for tuberculosis

The development of diagnostic tests which can readily differentiate between vaccinated and tuberculosis-infected individuals is crucial for the wider utilization of bacillus Calmette-Guérin (BCG) as vaccine in humans and animals. BCG_0092 is an antigen that elicits specific delayed type hypersensitivity reactions similar in size and morphological aspects to that elicited by purified protein derivative, in both animals and humans infected with the tubercle bacilli. We carried out bioinformatics analyses of the BCG_0092 and designed a diagnostic test by using the predicted MHC class I epitopes. In addition, we performed a knockout of this gene by homologous recombination in the BCG vaccine strain to allow differentiation of vaccinated from infected individuals. For that, the flanking sequences of the target gene (BCG_0092)were cloned into a suicide vector. Spontaneous double crossovers, which result in wild type revertants or knockouts were selected using SacB. BCG_0092 is present only in members of the Mycobacterium tuberculosis complex. Eight predicted MHC class I epitopes with potential for immunological diagnosis were defined, allowing the design of a specific diagnostic test. The strategy used to delete the (BCG_0092) gene from BCG was successful. The knockout genotype was confirmed by PCR and by Southern blot. The mutant BCG strain has the potential of inducing protection against tuberculosis without interfering with the diagnostic test based on the use of selected epitopes from BCG_0092.

Construction of a new vector for targeted gene disruption in Candida albicans / 中华传染病杂志

Objective To explore the feasibility of using Zeocin resistance as a new positive selection marker for the genetic manipulation of Candida albicans (C.albicans).Methods The susceptibility of C.albicans strain CAI4 to Zeocin was determined using a broth microdilution method in pH range from 6.0 to 8.0.The ZeoR expression cassette was amplified from plasmid pGAPZ-α-A by polymerase chain reaction (PCR).An integrated vector used to knockout AAF1 gene was constructed by gene splicing.Then the C.albicans CAI4 was transformed with this integrated plasmid containing the ZeoR expression cassette using lithium chloride method and the target AAF1 gene was replaced.The recombinant C.albicans was screened and the genome was extracted and amplified by PCR with two pairs of primers,then confirmed using genetic method.Results The C.albicans CAI4 was sensitive to Zeocin in pH range from 6.0 to 8.0.And the concentration of Zeoein(100 mg/L) at pH 7.0 was used to select recombined C.albicans.The recombined integrated vector was confirmed by DNA sequencing.The ZeoR expression cassette was detected using PCR method in recombined C.albicans.The AAF1 gene was replaced by ZeoR gene.Conclusion A new vector for targeted gene disruption in C.albicans is successfully constructed.