Development and application of a rapid gene manipulating toolbox for Pseudomonas aeruginosa / 生物工程学报

Manipulation of genes, including knock-out or knock-in, replacement of gene elements (such as promoters), fusion with a fluorescent protein gene, and construction of in situ gene reporter, is required in most of the biotechnological laboratories. The widely used gene manipulating methods based on two-step allelic exchange are cumbersome in terms of constructing plasmids, transforming and screening. In addition, the efficiency of using this method for long fragment knockout is low. To simplify the process of gene manipulation, we constructed a minimized integrative vector pln2. When a gene needs to be inactivated, an internal fragment of the target gene (non-frameshift) is cloned into the pln2 plasmid. Once the single-crossover recombination between genome and the constructed plasmid occurs, the endogenous gene is segmented by the plasmid backbone and thus inactivated. We developed a toolbox based on pln2 that can be used for different genomic operation mentioned above. With the help of this toolbox, we successfully knocked out large fragments of 20-270 kb.

Generation of Mlk3 KO mice by CRISPR/Cas9 and its effect on blood pressure / 生物工程学报

To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.

Berberine Inhibits Atherosclerosis by Regulating Lipophagy Via Targeting Wnt5a/NPC1 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the regulatory effect and molecular mechanism of berberine （BBR） on lipophagy in the prevention and treatment of atherosclerotic （AS） lesions in mice. MethodFifty apolipoprotein E-knockout （ApoE-/-） mice were randomly divided into an AS model group， an atorvastatin group （5 mg·kg-1）， and low-， medium-， and high-dose BBR groups （2.5， 5， 10 mg·kg-1）. Ten C57BL/6J mice were assigned to the control group. After 12 weeks， hematoxylin-eosin （HE） and oil red O staining were performed to assess the histopathological changes of AS plaques in the aorta. Biochemical analysis was used to measure serum lipid levels， and enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of inflammatory cytokines interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α）， oxidative stress marker reactive oxygen species （ROS）， and serum lipophagy marker Beclin1 and microtubule-associated protein 1 light chain 3 Ⅱ （LC3Ⅱ）. The xanthine oxidase method was used to measure serum superoxide dismutase （SOD） activity. Immunohistochemistry （IHC） was used to detect the distribution of wingless-type MMTV integration site family member 5a （Wnt5a） and Nieman Pick type C1 （NPC1） in the aorta， and Western blot was used to determine the protein expression of Wnt5a and NPC1 in the aorta. ResultCompared with the control group， the AS model group showed significant AS plaque formation， significantly elevated levels of serum total cholesterol （TC）， triglycerides （TG）， low-density lipoprotein cholesterol （LDL-C）， IL-6， TNF-α， and ROS， aortic Wnt5a distribution and protein expression （P<0.01）， and significantly reduced levels of serum high-density lipoprotein cholesterol （HDL-C）， SOD， Beclin1， LC3Ⅱ， and aortic NPC1 distribution and protein expression （P<0.01）. Compared with the AS model group， the atorvastatin group， and high- and medium-dose BBR groups showed a significant reduction in AS plaque area （P<0.05， P<0.01）， significantly decreased levels of serum TC， TG， LDL-C， IL-6， TNF-α， ROS， and aortic Wnt5a distribution and protein expression （P<0.05， P<0.01）， and significantly increased levels of serum HDL-C， SOD， Beclin1， LC3Ⅱ， and aortic NPC1 distribution and protein expression （P<0.05， P<0.01）. There was no statistically significant difference in the above indicators between the atorvastatin group and the medium-dose BBR group. ConclusionBBR can competitively bind to Wnt5a to activate NPC1 expression， upregulate lipophagy levels， reduce blood lipids， and inhibit the release of inflammatory mediators and oxidative stress damage， thereby exerting a preventive and therapeutic effect on AS.

Mechanism of Renshentang in Treatment of Atherosclerosis Based on Autophagic Effect of TRPV1 on Arterial Smooth Muscle / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Renshentang， recorded in Synopsis of Golden Chamber， in the treatment of atherosclerosis （AS） based on the autophagic effect of transient receptor potential vanilloid subtype 1 （TRPV1） on arterial smooth muscle. MethodFourteen SPF-grade 8-week-old male C57BL/6J mice were assigned to the normal group and 70 8-week-old apolipoprotein E knockout （ApoE-/-） mice were assigned to the experimental group. The AS model was induced by a high-fat diet in the mice in the experimental group for eight weeks. The model mice were then randomly divided into model group， low-， medium-， and high-dose Renshentang groups （2.715， 5.43， and 10.68 g·kg-1·d-1）， and simvastatin group （0.02 g·kg-1·d-1）. Drug treatment lasted eight weeks. Serum was taken and serum total cholesterol （CHO）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C）， and high-density lipoprotein cholesterol （HDL-C） levels were measured by assay kits to observe the changes in lipid levels in mice. The aorta was stained with hematoxylin-eosin （HE） to observe the overall pathology of the aortic root and oil red O staining was used to detect the lipid deposition in the aortic plaque and calculate the percentage of the aortic root area to the lumen area. The protein expression of TRPV1， adenylate-activated protein kinase （AMPK）， phosphorylated AMPK （p-AMPK）， autophagy effector-1 （Beclin-1）， and microtubule-associated protein 1 light chain 3 （LC3Ⅱ） in mouse aortic tissues was determined by Western blot. ResultCompared with the normal group， the model group showed increased serum CHO， TG， and LDL-C levels， decreased HDL-C， and increased aortic root plaque area （P<0.01）. Compared with the model group， the Renshentang groups showed decreased levels of CHO， TG， and LDL-C in serum （P<0.05， P<0.01）， especially in the low- and medium-dose Renshentang groups （P<0.01）. Compared with the normal group， the simvastatin group and the Renshentang groups showed reduced aortic root plaque area （P<0.05）， especially in the high-dose Renshentang group （P<0.01）. Compared with the normal group， the model group showed decreased relative expression levels of TRPV1， p-AMPK/AMPK， Beclin-1， and LC3Ⅱ/LC3Ⅰ（P<0.05， P<0.01）. Compared with the model group， the medium- and high-dose Renshentang groups showed increased relative expression levels of TRPV1， p-AMPK/AMPK， Beclin-1， and LC3Ⅱ/LC3Ⅰ（P<0.05，P<0.01）. ConclusionThe anti-AS effect of Renshentang recorded in Synopsis of Golden Chamber may be achieved by up-regulating TRPV1 expression to restore the level of autophagy mediated by AMPK.

Macrophage MED1 deficiency promotes the development of atherosclerosis in female ApoE and LDLR knockout mice / 西安交通大学学报(医学版)

【Objective】 To study the effect of macrophage mediator 1 (MED1) deficiency on atherosclerosis in female mice. 【Methods】 ApoE knockout (ApoE-/-), LDLR knockout (LDLR-/-), MED1fl/fl, and macrophage MED1 knockout (MED1△Mac) mice were recruited in the study. Two types of mouse model were constructed:ApoE and macrophage MED1 double knockout (MED1△Mac/ApoE-/-) mice and their littermate controls (MED1fl/fl/ApoE-/-). ② LDLR knockout (LDLR-/-) mice receiving bone marrow from MED1△Mac (MED1△Mac→LDLR-/-) or MED1fl/fl (MED1fl/fl→LDLR-/-) mice. Female mice from these two models were fed a Western diet (21% fat and 0.15% cholesterol) for 12 weeks to promote the development of atherosclerosis. Body weight, total cholesterol (TC), and total triglyceride (TG) content in plasma were measured dynamically. After Western diet feeding for 12 weeks, aortic tree and aortic root were collected and hematoxylin-eosin (H&E) and oil red O staining were performed. 【Results】 Plasma TC and TG did not significantly differ between MED1fl/fl/ApoE-/- control group and MED1△Mac/ApoE-/-experimental group. However, the plaque area in aortic tree and aortic root was significantly increased in MED1△Mac/ApoE-/-mice. Moreover, compared with that in MED1fl/fl→LDLR-/- control group, the plaque area of aortic tree and aortic root had an increasing trend in MED1△Mac→LDLR-/- mice group. 【Conclusion】 MED1 deficiency in macrophages promotes the development of atherosclerosis in female ApoE or LDLR knockout mice.

Effect of intestinal Metrnl gene knockout on intestinal microbiota and ulcerative colitis / 药学实践杂志

Objective To investigate the effect of intestinal Metrnl on dextran sodium sulfate (DSS)-induced ulcerative colitis mouse model and the regulation mechanism of intestinal microbiota. Methods Different concentrations of DSS (3% DSS and 1% DSS) were used to induce ulcerative colitis on C57 mice to determine the experimental conditions. Intestinal epithelial Metrnl specific knockout mice (Metrnl(-/-)) and its control mice (Metrnl(+/+)) were administrated with 3% DSS for 5 d. Then the survival time, body weight, DAI (disease activity index), colon length and pathological changes in colon tissues were observed. 16S ribosomal RNA gene sequencing was used to detect the composition of intestinal microbiota. Results Compared with 1% DSS, 3% DSS could significantly aggravate ulcerative colitis on C57 mice, such as lower survival rate (P<0.05), more weight loss (P<0.05), higher DAI score (P<0.05), shorter colon length (P<0.05) and higher pathology score (P<0.05). After administrated to 3% DSS for 5 d, comparing with Metrnl(+/+) mice, Metrnl(-/-) mice showed more weight loss (P<0.05), higher DAI score (P<0.05), shorter colon length (P<0.05) and higher pathology score (P<0.05). The 16S ribosomal RNA results showed that the diversity of intestinal microbiota in Metrnl(-/-) mice significantly decreased. Furthermore, Bacteroidetes and Proteobacteria significantly decreased, while Firmicutes increased. Conclusion Metrnl could protect the DSS-induced ulcerative colitis mouse through regulating intestinal microbiota.

Effect of ACE2 deletion on vasoconstriction reactivity of aortic segments in mice with tourniquet shock / 中国病理生理杂志

AIM:To observe the effect of angiotensin-converting enzyme 2(ACE2)deletion on vasoconstric-tion reactivity of aortic segments in ACE2 knockout(KO)mice with tourniquet shock(TS).METHODS:The 8-month-old male mice with C57BL/6 background were divided into wild-type(WT)control group,WT-TS group,KO group and KO-TS group,with 10 mice in each group,of which five were used for determination of vascular reactivity,and the other five for the other assays.The hindlimbs of the mice in WT-TS group and KO-TS group were ligated with tourniquet for 2 h and loosened for 4 h.The mice in WT group and KO group were subjected to the same treatment except for tourniquet liga-tion.The vasoconstriction reactivity of the aorta was measured on tensiometer.The morphological damage of the aorta was evaluated by vascular histopathology.Western blot was used to detect the expression of AT1,MAS,ACE and ACE2 pro-teins in aorta.The serum levels of angiotensin(Ang)Ⅱ and Ang-(1-7)were determined by enzyme-linked immunosorbent assay.RESULTS:Compared with WT group,the mice in WT-TS group had lower vascular reactivity to norepinephrine(NE)and obvious vascular lesions.The expression of ACE protein increased significantly(P＜0.01),while the expres-sion of ACE2 decreased(P＜0.05).The expression of AT1 protein in aorta decreased significantly,the expression of MAS protein increased significantly,and the AT1/MAS ratio decreased(P＜0.01).Serum Ang II level increased,serum Ang-(1-7)level decreased,and Ang Ⅱ/Ang-(1-7)ratio increased(P＜0.05).Compared with WT group,vascular reactivity in KO group increased at low concentration of NE(＜10-7 mol/L),and decreased at high concentration(＞10-7 mol/L)without vascular lesion.The expression levels of aortic AT1,MAS and ACE were all elevated(P＜0.05).The serum level of Ang Ⅱ increased(P＜0.05),but the level of Ang-(1-7)had no obvious change.Compared with KO and WT-TS groups,the aortic reactivity in KO-TS group subtracted apparently(P＜0.05),representing its curve shifting to the right obviously.The morphological damage aggravated slightly,and the expression of AT1 and ACE increased slightly in KO-TS group com-pared with WT-TS group(P＜0.05).However,the expression of MAS increased significantly in vascular tissue(P＜0.01).The serum levels of Ang Ⅱ and Ang-(1-7)further increased and decreased,respectively,and the Ang Ⅱ/Ang-(1-7)ratio increased(P＜0.01).CONCLUSION:Deficiency of ACE2 induces severe aortic hyporeactivity to NE during TS,which may be related to the increased imbalance of renin-angiotensin system in ACE2 gene knockout mice.

Síndrome de Baller-Gerold. A propósito de un caso clínico y revisión de la literatura

El síndrome de Baller-Gerold es secundario a mutaciones en el gen RECQL4 (8q24.3). Este gen pertenece a la familia de las RecQhelicasas y está implicado en otras enfermedades de predisposición al cáncer. El diagnóstico se basa en criterios clínicos y debido al elevado número de diagnósticos diferenciales, encontrar una mutación en el gen puede ayudar a precisar el espectro diagnóstico, el consejo genético y su tratamiento. Se han descrito en la literatura alrededor de 30 casos, aunque se sabe que se ha presentado en menos de 200 000 personas en el mundo, por lo que se considera, una condición clínica rara. Se presenta el caso de una paciente que desde su nacimiento se constataron múltiples malformaciones músculo-esqueléticas: aplasia radial, pulgares aplásicos, malformaciones de la parrilla costal, clinodactilia de todos los dedos de miembros superiores, antebrazos hipoplásicos, clinodactilia del miembro inferior izquierdo. Se le realizó alimentación parenteral por varios días, fue imposible establecer vía oral adecuada por ausencia y/o incoordinación de los reflejos de succión-deglución como expresión de sus malformaciones a nivel de sistema nervioso central. Se presenta el caso por su baja incidencia y prevalencia por lo que es considerada una enfermedad rara.

Baller-Gerold syndrome is secondary to mutations in the RECQL4 gene (8q24.3). This gene belongs to the RecQhelicase family and is implicated in other diseases predisposing to cancer. Diagnosis is based on clinical criteria and due to the high number of differential diagnoses, finding a mutation in the gene can help to specify the diagnostic spectrum, genetic counseling and treatment. Around 30 cases have been described in the literature, although it is known that it has occurred in less than 200,000 people in the world, being considered a rare clinical condition. We present the case of a newborn, who at birth was found to have multiple musculoskeletal malformations: radial aplasia, aplastic thumbs, malformations of the rib cage, clinodactyly of all the fingers of the upper limbs, hypoplastic forearms, clinodactyly of the left lower limb. She was given parenteral feeding for several days, it was impossible to establish an adequate oral route due to the absence and/or incoordination of the sucking-swallowing reflexes as an expression of her malformations at the central nervous system level. The case is presented due to its low incidence and prevalence, which is why it is considered a rare disease.

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.

Intervention of Huanglian Jiedutang on Atherosclerosis in ApoE-/- Mice and Its Mechanism / 中国实验方剂学杂志

ObjectiveTo study the intervention of Huanglian Jiedutang on atherosclerosis （AS） in apolipoprotein E knockout （ApoE-/-） mice induced by the high-fat diet. MethodThe ApoE-/- mouse model of AS was induced by the high-fat diet， and Huanglian Jiedutang was used to intervene in the AS in the ApoE-/- mice. The pathological changes of aorta were observed by hematoxylin-eosin （HE） staining. The levels of serum total cholesterol （TC）， triglyceride （TG）， high-density lipoprotein cholesterol （HDL-C）， and low-density lipoprotein cholesterol （LDL-C） were detected by an automatic biochemical analyzer. The protein expression levels of sirtuin-1 （SIRT1） and nuclear factor-kappa B （NF-κB） were determined by Western blot assay， and the mRNA expression levels of adenosine 5'-monophosphate-activated protein kinase （AMPK）， peroxisome proliferators-activated receptors α （PPARα）， tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， interleukin-1β （IL-1β）， and NOD-like receptor pyrin domain-containing 3 （NLRP3） were determined by real-time quantitative polymerase chain reaction （Real-time PCR）. ResultAs compared with the normal group， there was a large amount of lipid accumulation in the blood vessels of the model group. In the model group， the levels of serum TG， TC， and LDL-C were increased （P<0.01）， and the level of HDL-C was decreased （P<0.01）. The protein expression level of SIRT1 in the aorta was decreased， while that of NF-κB was increased in the model group （P<0.01）. The mRNA expression levels of IL-6， TNF-α， and IL-1β were higher （P<0.01）， while those of AMPK in the liver were lower in the model group （P<0.01）. Compared with the model group， the Huanglian Jiedutang group reduced the lipid accumulation and inflammatory reaction in the aorta of mice with AS， reduced the levels of TC， TG， and LDL-C （P<0.01）， and increased the level of HDL-C （P<0.01）. Huanglian Jiedutang significantly increased the protein expression level of SIRT1 in the aorta of ApoE-/- mice （P<0.01） and decreased the protein expression levels of NF-κB in the aorta （P<0.05， P<0.01）. Huanglian Jiedutang down-regulated the mRNA expression levels of TNF-α， IL-6， IL-1β， and NLRP3 in the aorta （P<0.05， P<0.01）， and up-regulated the mRNA expression levels of AMPK and PPARα in the liver of ApoE-/- mice （P<0.05， P<0.01）. ConclusionHuanglian Jiedutang has a certain intervention effect on the formation of atherosclerotic aortic plaque in ApoE-/- mice. Its mechanism may be related to the decrease of serum TC， TG， and LDL-C levels， the increase of HDL-C levels， thus playing a role in lowering blood lipid， the increase of SIRT1 protein， the decrease of NF-κB protein， the decrease of inflammatory factors such as TNF-α and IL-6， which protects blood vessels from inflammatory injury， and the improvement of AMPK and PPARα levels to participate in autophagy and apoptosis.

Impact of angiotensin-converting enzyme inhibition on hemodynamic and autonomic profile of elastase-2 knockout mice

Elastase-2 (ELA-2) is an angiotensin II-generating enzyme that participates in the cardiovascular system. ELA-2 is involved in hemodynamic and autonomic control and is upregulated in myocardial infarction and hypertension. The inhibition of angiotensin-converting enzyme (ACE) increased ELA-2 expression in the carotid arteries and heart of spontaneously hypertensive rats. In this study, we sought to investigate the role of ACE inhibition in hemodynamic and autonomic balance in elastase-2 knockout (ELA-2 KO) mice. Male ELA-2 KO and C57BL/6 mice were treated with the ACE inhibitor enalapril or saline for 10 days. After treatment, mice underwent surgery for cannulation of the femoral artery and arterial pressure recordings were made five days later in awake animals. The variability of systolic blood pressure (SBP) and pulse interval (PI) was evaluated in the time and frequency domain. Spontaneous baroreflex was assessed by the sequencing method. ACE inhibition caused a significant decrease in mean arterial pressure (117±2.2 vs 100±2.8 mmHg) and an increase in heart rate (570±32 vs 655±15 bpm) in ELA-2 KO mice. Despite a tendency towards reduction in the overall heart rate variability (standard deviation of successive values: 7.6±1.1 vs 4.7±0.6 ms, P=0.08), no changes were found in the root of the mean sum of squares or in the power of the high-frequency band. ACE inhibition did not change the spontaneous baroreflex indices (gain and baroreflex effectiveness index) in ELA-2 KO mice. Altogether, this data suggested that ACE played a role in the maintenance of hemodynamic function in ELA-2 KO mice.

Effects of liver-specific Nampt knockout on ischemic stroke / 药学实践杂志

Objective Nicotinamide phosphoribosyltransferase (Nampt) is a new therapeutic target for ischemic stroke. The aim of this study was to investigate protective effect of liver-derived Nampt on ischemic stroke. Methods Liver-specific Nampt knockout mice were generated using the Cre/loxP system. NamptloxP/loxP mice were crossed with liver-specific Cre recombinase expression mice (Alb-Cre), and the progeny genotypes were identified by polymerase chain reaction. Body weight of knockout mice and control mice were measured. Nampt in liver and brain was determined by Western blot assay. Middle cerebral artery occlusion (MCAO), a classical ischemic stroke model, was generated in liver-specific Nampt knockout mice and control mice by electrocoagulation. After 24 h of modeling, neurological deficit scores of each group were evaluated and TTC staining was performed to determine the cerebral infarction volume. The level of plasma Nampt in each group was determined by ELISA. Results Liver-specific Nampt knockout mice with the genotype of NamptloxP/loxPAlb-Cre were successfully constructed. The hepatic Nampt expression in knockout mice was significantly decreased by 74.2% compared to control mice, while there was no significant difference in the expression of brain Nampt protein between the knockout group and the control group. Specific knockout of liver Nampt gene expression had no effect on the body weight of mice. Under normal physiological conditions, there was no significant difference in plasma Nampt levels between liver-specific Nampt knockout mice and control mice of the same gender. 24 h after MCAO modeling, there were no significant differences in neurological deficit scores, cerebral infarct volume and plasma Nampt concentration between liver-specific Nampt knockout group and control group. Conclusion Liver-specific Nampt knockout mice are successfully constructed. Liver-derived Nampt has no significant protective effects on ischemic stroke.

Effects of Bcl3 gene knockout on composition of spleen immune cells and antitumor ability of mice / 中华微生物学和免疫学杂志

Objective:To investigate the effects of Bcl3 gene knockout on the composition of spleen immune cells and antitumor ability of mice.Methods:Bcl3 gene knockout mice (Bcl3 -/-) were established by CRISPR/Cas9 genome editing technology. Blood routine test and flow cytometry were used to detect the immune cell composition in Bcl3 -/- mice. Lung metastasis models were established by injecting mice with B16F10 melanoma cells. The number of tumor nodules in lung and the survival time of mice were used to assess the antitumor ability of wild-type (WT) and Bcl3 -/- mice. Results:Bcl3 -/- mice were successfully bred to a strain with normal growth rate and normal breeding performance. Furthermore, no embryonic death occurred. Compared with WT mice, Bcl3 -/- mice showed splenomegaly and a significant increase in the number of spleen immune cells ( P<0.05). The counts and percentages of platelets and neutrophils in Bcl3 -/- mice were significantly lower than those in WT mice. The proportion of CD19 + B cells showed no significant change, while the proportions of CD3 + T cells and T cell subsets (CD4 + , CD8 + , Treg) increased significantly ( P<0.05). The proportions of NK cells (NK1.1 + ) and neutrophils (Gr1 + ) decreased ( P<0.05), while no significant change in the proportion of DC (CD11b + ) was observed. There were a large number of tumor nodules formed by melanoma cells in the lung of Bcl3 -/- tumor bearing mice, and their survival time was shortened dramatically. Conclusions:Knockout of Bcl3 gene affected the development, differentiation and function of immune cells, thereby reducing the antitumor ability of mice.

Construction of a Spink5 conditional knockout mouse model and analysis of its phenotype / 中华皮肤科杂志

Objective:To construct a serine protease inhibitor Kazal type-5 (Spink5) conditional knockout mouse model, and to identify its phenotype.Methods:B cell-specific Spink5 conditional knockout mice of genotype Mb1 cre/+Spink5 floxp/floxp were constructed by using clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated protein 9 (Cas9) technology, and served as the knockout group. Mice of genotype Mb1 +/+Spink5 floxp/floxp served as the control group. The mice of genotype Mb1 cre/+Spink5 floxp/floxp or Mb1 +/+Spink5 floxp/floxp were sacrificed when they were 4 to 6 weeks old, splenic mononuclear cells were isolated, and B lymphocytes and non-B lymphocytes were sorted by flow cytometry and fluorescence-activated cell sorting. Genotype identification was performed by PCR, and protein expression of lymphoepithelial Kazal-type-related inhibitor (LEKTI) was determined by Western blot analysis. Skin tissues were resected from the mice, and subjected to hematoxylin-eosin staining for measuring the epidermal thickness. Immunofluorescence staining was performed to determine fluorescence intensity of LEKTI protein in the mouse skin tissues. Paired t test or two-independent-sample t test was used for comparisons between groups. Results:Genotype identification results demonstrated that the stable B lymphocyte-specific Spink5 conditional knockout mouse model was successfully constructed. Western blot analysis revealed that the relative protein expression of LEKTI in the B lymphocytes in the knockout group was 0.01 ± 0.02, which was significantly lower than that in the non-B lymphocytes in the knockout group (0.66 ± 0.11, t = 9.99, P < 0.001) , and that in the B lymphocytes in the control group (1.08 ± 0.13, t = 13.78, P < 0.001) . Among 39 mice in the knockout group, 4 presented with dry skin and scattered scaly hypertrophic maculopapules. The epidermal thickness of the lesional skin tissues in the knockout group was 90.42 ± 21.31 μm, significantly higher than that of the non-lesional skin tissues in the knockout group (29.71 ± 3.63 μm, t = 5.05, P = 0.002) and that of normal skin tissues in the control group (12.42 ± 2.21 μm, t = 6.74, P < 0.001) . Immunofluorescence staining showed no significant difference in the fluorescence intensity of LEKTI protein among the lesional skin tissues (46.21 ± 1.21) , non-lesional skin tissues (46.62 ± 2.13) in the knockout group and normal skin tissues in the control group (47.69 ± 1.71, P > 0.05) . Conclusion:The B lymphocyte-specific Spink5 conditional knockout mouse model was successfully constructed, which provides a basis for further exploring mechanisms underlying skin barrier defects and immune dysfunction in Netherton syndrome.

Effect of Ethyl Acetate Extract of Acanthopanacis Senticosi Radix et Rhizoma seu Caulis on Intestinal Flora in ApoE-/- Mice with Atherosclerosis / 中国实验方剂学杂志

ObjectiveTo observe the effect of ethyl acetate extract of Acanthopanacis Senticosi Radix et Rhizoma seu Caulis on high-fat diet-induced apolipoprotein E gene knockout （ApoE-/-） mice， and explore its mechanism of treating atherosclerosis by regulating intestinal flora. MethodThirty-two 8-week-old male ApoE-/- mice were randomly divided into model group， rosuvastatin group （10 mg·kg-1）， high-， low-dose groups of ethyl acetate extract of Acanthopanacis Senticosi Radix et Rhizoma seu Caulis （75， 25 mg·kg-1）， with 8 mice in each group. Eight C57BL/6 mice were used as blank group. After 8 weeks of continuous administration， blood was taken to determine the blood lipid level. Enzyme-linked immunosorbent assay （ELISA） was used to detect the contents of related indexes in serum of mice. Hematoxylin-eosin （HE） staining was used to observe the formation of aortic plaque in mice. Cecal contents were collected and 16S rRNA amplicon sequencing was used to detect intestinal flora. ResultCompared with the blank group， the plaque area of the model group was significantly increased with inflammatory infiltration， the contents of triglyceride （TG）， total cholesterol （TC）， low-density lipoprotein cholesterol （LDL-C）， inflammatory factors and inducible nitric oxide synthase （iNOS） were increased， while the content of high-density lipoprotein cholesterol （HDL-C） was decreased. Compared with the model group， rosuvastatin group and high- and low-dose groups of ethyl acetate extract of Acanthopanacis Senticosi Radix et Rhizoma seu Caulis could improve the deposition of aortic plaque， reduce the contents of TG， TC， LDL-C， inflammatory factors and iNOS， and increase the content of HDL-C. Compared with the blank group， the relative abundances of Firmicutes and Proteobacteria in the model group increased， while the relative abundance of Bacteroidetes decreased. Alpha and Beta diversity analysis showed that samples of each group could be significantly isolated， and the total number and abundance of intestinal flora species in the model group were low. Compared with the model group， ethyl acetate extract of Acanthopanacis Senticosi Radix et Rhizoma seu Caulis could increase the relative abundance of beneficial bacteria and decrease the relative abundance of pathogenic bacteria. ConclusionEthyl acetate extract of Acanthopanacis Senticosi Radix et Rhizoma seu Caulis was mainly composed of flavonoids， which can treat atherosclerosis by regulating the intestinal flora and improve the pathological changes in the aorta of ApoE-/- mice induced by high-fat diet. The mechanism may be related to its ability to reduce the level of inflammatory factors， improve antioxidant capacity and repair the disorder of intestinal flora structure.

Effect and Mechanism of Longshengzhi Capsule on Atherosclerosis in ApoE-/- Mice / 中国实验方剂学杂志

ObjectiveTo study the effect of Longshengzhi capsule （LSZC） on high fat diet （HFD）-induced atherosclerosis （AS） in apolipoprotein E knockout （ApoE-/-） mice. MethodApoE-/- mice were fed with HFD for 8 weeks to induce AS. Then the mice were randomized into model group， simvastatin group （4 mg·kg-1）， high-dose LSZC group （1.6 g·kg-1）， medium-dose LSZC group （0.8 g·kg-1）， and low-dose LSZC group （0.4 g·kg-1）. C57BL/6J Mice with normal diet were used as the blank control. After 10 weeks， serum levels of triglyceride （TG）， total cholesterol （TC）， high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， malondialdehyde （MDA）， superoxide dismutase （SOD）， interleukin-1β （IL-1β）， and interleukin-6 （IL-6） were detected. Hematoxylin-eosin （HE） and oil red O were used to detect aortic plaque in each group. The levels of CD34 and F4/80 in aorta were determined by immunohistochemistry （IHC）. ResultCompared with the blank control， the model group demonstrated obvious aortic plaque， a large amount of lipid accumulation， serious damage of aortic intima， increase in serum levels of TC， TG， LDL-C， HDL-C， MDA， IL-1β， and IL-6 （P<0.01）， decrease in SOD level （P<0.01）， and rise of the expression of CD34 and F4/80 （P<0.01）. Compared with the model group， LSZC of the three doses all decreased the serum levels of TG and LDL-C （P<0.05）， and the levels of IL-1β and IL-6 （P<0.05， P<0.01）， and the high-dose and medium-dose LSZC improved SOD level， decreased MDA content （P<0.05， P<0.01）， and reduced the expression of the CD34 and F4/80 in blood vessels （P<0.05， P<0.01）. ConclusionLSZC has certain intervention effect on the formation of aortic plaque in atherosclerosis ApoE-/- mice. The mechanism is that it reduces the levels of serum TG and LDL-C to lower blood lipid， decreases MDA level and improves SOD activity to inhibit lipid peroxidation， lowers the levels of IL-1β and IL-6 and down-regulates the expression of CD34 and F4/80 to protect blood vessels from inflammatory damage.

Knockout of farnesoid X receptor gene aggravates cisplatin-induced kidney injury / 中南大学学报(医学版)

OBJECTIVES@#Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily of ligand activated transcription factors and belongs to bile acid receptor. Studies have shown that the expression of FXR in renal tissue can reduce renal injury via regulation of glucose and lipid metabolism, inhibition of inflammatory response, reduction of oxidative stress and renal fibrosis. However, it is unclear whether FXR is involved in autophagy in renal diseases. This study aims to investigate the role of FXR in cisplatin-induced acute renal injury and whether its mechanism is related to autophagy regulation.@*METHODS@#Twelve male WT or FXR-KO mice at 12 weeks were randomly divided into a WT group, a WT+cisplatin group, a FXR-KO group, and a FXR-KO+cisplatin group, with 6 mice in each group. The WT+cisplatin group and the FXR-KO+cisplatin group were intraperitoneally injected with cisplatin (20 mg/kg), and the WT group and the FXR-KO group were intraperitoneally injected with equal volume of cisplatin solvent. Seventy-two hours later, the mice were killed and blood and renal tissue samples were collected. The levels of SCr and BUN were detected by immunoturbidimetry. After the staining, the pathological changes of renal tissue were observed under optical microscope. The protein levels of LC3 and p62 were detected by Western blotting and immunohistochemistry. The clearance of damaged mitochondria and the accumulation of lysosomal substrate were observed under electron microscope. The apoptosis of renal tubular epithelial cells was detected by TUNEL.@*RESULTS@#Compared with the WT group or the FXR-KO group, both SCr and BUN levels in the WT+cisplatin group or the FXR-KO+cisplatin group were significantly increased (P<0.01 or P<0.001), and SCr and BUN levels in the FXR-KO+cisplatin group were significantly higher than those in the WT+cisplatin group (both P<0.05). Under the light microscope, there were no obvious pathological changes in the renal tissue of mice in the WT group and the FXR-KO group. Both the WT+cisplatin group and the FXR-KO+cisplatin group had vacuolar or granular degeneration of renal tubular epithelial cells, flat cells, lumen expansion, brush edge falling off, and even exposed basement membrane and tubular formation. The scores of renal tubular injury in the WT+cisplatin group and the FXR-KO+cisplatin group were significantly higher than those in the WT group and the FXR-KO group, respectively (both P<0.001), and the score in the FXR-KO+cisplatin group was significantly higher than that in the WT+cisplatin group (P<0.05). Under the transmission electron microscope, the mitochondria of mouse tubular epithelial cell in the WT+cisplatin group and the FXR-KO+cisplatin group was swollen, round, vacuolated, cristae broken or disappeared; the lysosome was uneven and high-density clumps, and the change was more obvious in the FXR-KO+cisplatin group. Western blotting showed that the ratio of LC3-II to LC3-I was decreased and the expression of p62 was increased in the WT+cisplatin group compared with the WT group and the FXR-KO+cisplatin group compared with FXR-KO group (P<0.05 or P<0.01); compared with the FXR-KO group, the ratio of LC3-II to LC3-I was decreased and the expression of p62 was increased significantly in the FXR-KO+cisplatin group (both P<0.05). Immunohistochemistry results showed that the expression of total LC3 and p62 in renal cortex of the WT+cisplatin group and the FXR-KO+cisplatin group was increased significantly, especially in the FXR-KO+cisplatin group. TUNEL results showed that the mice in the WT group and the FXR-KO group had negative staining or only a few apoptotic tubular epithelial cells, and the number of apoptotic cells in the WT+cisplatin group and the FXR-KO+cisplatin group were increased. The apoptosis rates of renal tubular epithelial cells in the WT+cisplatin group and the FXR-KO+cisplatin group were significantly higher than those in the WT group and the FXR-KO group, respectively (both P<0.001), and the apoptosis rate in the FXR-KO+cisplatin group was significantly higher than that in the WT+cisplatin group (P<0.05).@*CONCLUSIONS@#Knockout of FXR gene aggravates cisplatin induced acute renal injury, and its mechanism may be related to inhibiting autophagy and promoting apoptosis.

CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice / 亚洲男科学杂志(英文版)

Gene expression analyses suggest that more than 1000-2000 genes are expressed predominantly in mouse and human testes. Although functional analyses of hundreds of these genes have been performed, there are still many testis-enriched genes whose functions remain unexplored. Analyzing gene function using knockout (KO) mice is a powerful tool to discern if the gene of interest is essential for sperm formation, function, and male fertility in vivo. In this study, we generated KO mice for 12 testis-enriched genes, 1700057G04Rik, 4921539E11Rik, 4930558C23Rik, Cby2, Ldhal6b, Rasef, Slc25a2, Slc25a41, Smim8, Smim9, Tmem210, and Tomm20l, using the clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9 (CRISPR/Cas9) system. We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation. Mating tests of KO mice reveal that these 12 genes are not essential for male fertility, at least when individually ablated, and not together with other potentially compensatory paralogous genes. Our results could prevent other laboratories from expending duplicative effort generating KO mice, for which no apparent phenotype exists.

Effects of deleting peptidoglycan hydrolase genes on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease / 生物工程学报

In order to explore the effect of peptidoglycan hydrolase on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease, five peptidoglycan hydrolase genes (lytC, lytD, lytE, lytF and lytG) of B. amyloliquefaciens TCCC111018 were knocked out individually. The viable cell counts of the bacteria and their alkaline protease activities before and after gene deletion were determined. The viable cell counts of the knockout mutants BA ΔlytC and BA ΔlytE achieved 1.67×106 CFU/mL and 1.44×106 CFU/mL respectively after cultivation for 60 h, which were 32.5% and 14.3% higher than that of the control strain BA Δupp. Their alkaline protease activities reached 20 264 U/mL and 17 265 U/mL, respectively, which were 43.1% and 27.3% higher than that of the control strain. The results showed that deleting some of the peptidoglycan hydrolase genes effectively maintained the viable cell counts of bacteria and increased the activity of extracellular enzymes, which may provide a new idea for optimization of the microbial host for production of industrial enzymes.

Generation of genetic modified pigs devoid of GGTA1 and expressing the human leukocyte antigen-G5 / 生物工程学报

Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings. However, antibody-mediated immunity, which includes both natural and induced antibody responses, is a major challenge for the success of pig-to-primate xenotransplantation. Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation. In this study, we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1, 3-galactosyltransferase gene GGTA1, which encodes Gal epitopes that induce hyperacute immune rejection in pig-to-human xenotransplantation. Meanwhile, human leukocyte antigen-G5 gene HLA-G5, which acts as an immunosuppressive factor, was co-transfected with TALEN into porcine fetal fibroblasts. The cell colonies of GGTA1 biallelic knockout with positive transgene for HLA-G5 were chosen as nuclear donors to generate genetic modified piglets through a single round of somatic cell nuclear transfer. As a result, we successfully obtained 20 modified piglets that were positive for GGTA1 knockout (GTKO) and half of them expressed the HLA-G5 protein. Gal epitopes on the cell membrane of GTKO/HLA-G5 piglets were completely absent. Western blotting and immunofluorescence showed that HLA-G5 was expressed in the modified piglets. Functionally, the fibroblasts from the GTKO/HLA-G5 piglets showed enhanced resistance to complement-mediated lysis ability compared with those from GTKO-only or wild-type pigs. These results indicate that the GTKO/HLA-G5 pigs could be a valuable donor model to facilitate laboratory studies and clinics for xenotransplantation.