BACKGROUND:Solute carrier family 1 member 5(SLC1A5)plays a potential role in a variety of diseases,but the exact mechanism of action is unclear.The construction of stable SLC1A5 overexpression and knockdown cell models can provide a powerful experimental tool for in-depth study of the exact role and mechanism of SLC1A5 in diseases and the discovery of potential therapeutic targets. OBJECTIVE:To construct lentiviral vectors for overexpression and knockdown of mouse SLC1A5 and establish stable transfected RAW264.7 cell lines,so as to provide an experimental foundation for further investigation of the role of SLC1A5 in inflammation. METHODS:Primers were designed and synthesized based on the SLC1A5 gene sequence,and the gene segment was amplified using polymerase chain reaction.Subsequently,the target gene segment was directionally inserted into the GV492 vector plasmid,which had been digested with AgeI/NheI enzymes,to construct recombinant lentiviral plasmids.Positive clones were further selected,and their sequences were confirmed.The pHelper1.0 plasmid vector and pHelper2.0 plasmid vector,along with the target plasmid vector,was co-cultured with 293T cells for transfection,resulting in the production and titration of lentiviral stocks.Furthermore,RAW264.7 cells were cultured in vitro,and the working concentration of puromycin was determined.Lentiviruses were separately co-cultured with RAW264.7 cells,and transfection efficiency was determined by measuring fluorescence intensity.Stable transfected cells were selected using puromycin,and real-time fluorescence quantitative PCR and western blot assay were employed to assess the gene and protein expression levels of SLC1A5 in stably transfected cell lines. RESULTS AND CONCLUSION:(1)Sequencing results indicated a perfect match between the sequencing and target sequences,confirming the successful construction of recombinant lentiviral vectors.(2)The titer for the overexpression SLC1A5 lentivirus was 1×109 TU/mL,while the titer for the knockdown SLC1A5 lentivirus was 3×109 TU/mL.(3)The working concentration of puromycin for RAW264.7 cells was determined to be 3 μg/mL.(4)The optimal conditions for transfecting RAW264.7 cells with overexpression/knockdown expression of SLC1A5 lentivirus involved the use of HiTransG P transfection enhancer with a multiplicity of infection value of 50.(5)A significant upregulation of the gene and protein expression levels of SLC1A5 was detected in cell lines stably overexpressing SLC1A5,while gene and protein expression levels of SLC1A5 were significantly decreased in the knockdown stable cell lines.These findings indicate that lentiviral vectors for mouse SLC1A5 overexpression and knockdown have been successfully constructed and a stably transfected RAW264.7 cell line has been obtained.

Objective To overcome the limitations of existing human respiratory syncytial virus(hRSV)animal models,such as semi-permissiveness and short duration of infection,this study established an IL2rg gene knockout(IL2rg-/-)rat model using TALEN gene editing technology.Methods The animal model was infected with hRSV intranasally.Clinical characteristics,body weight,and temperature changes were observed over the infection period(0～35 days).The total viral loads in respiratory organs,such as the nasal tissue,trachea,and lungs,were measured at various time points(4,11,20,and 35 days post-infection).Pathological analysis was conducted on target organs at the endpoint of observation(35 days post-infection).Changes in peripheral blood T,B,NK,and NKT cells and various cytokines were assessed at various time points(4,20,and 35 days post-infection).Results(1)IL2rg/-knockout rats sustained high viral loads in the nasal cavity upon intranasal inoculation with hRSV.The average peak titer rapidly reached 1 × 1010 copies/g in nasal tissue and 1 × 107 copies/g up to 5 weeks post-infection.(2)However,no significant pathological changes were noted in nasal,tracheal,or lung tissues.(3)An increase was observed in the content of peripheral blood B cells in hRSV-infected IL2rg--rats.(4)IL-6 and MCP-1 were increased in the early stage of infection and then decreased at the end of the observation period.Conclusions This study established a new IL2rg-/-rat model using TALEN technology and found that this model effectively supported high-level replication and long-term infection of hRSV,providing a good basis for antiviral drug screening and in vivo efficacy evaluation of anti-hRSV antibodies.

OBJECTIVE To evaluate the mechanisms underlying the antidepressant effect of ZBH2012001,a novel serotonin and norepinephrine reuptake inhibitor(SNRI),in general and its ability to enhance monoaminergic transmission and suppress neuroinflammation in particular.METHODS① Male ICR mice were divided into vehicle(distilled water),duloxetine(DLX,10 or 20 mg·kg-1)and ZBH2012001(5,10 and 20 mg·kg-1)groups.One hour following ig administration,the antidepressant effect of ZBH2012001 was evaluated using the tail suspension test(TST)and forced swimming test(FST).② Radioligand binding assay was conducted to evaluate the affinity of ZBH2012001 for human serotonin transporters(hSERTs)and human norepinephrine transporters(hNETs).③ Mice were divided into vehicle(distilled water),DLX(10 or 20 mg·kg-1)and ZBH2012001(5,10 and 20 mg·kg-1)groups.One hour following drug administration,the 5-hydroxytryptophan(5-HTP)-induced head-twitch test or yohimbine-induced lethality test were performed to evaluate the effect of ZBH2012001 on the function of the 5-hydroxytryptamine(5-HT)and norepinephrine(NE)systems.④ Mice were divided into vehicle(distilled water+0.1%acetic acid),reserpine model(distilled water+reserpine 5 mg·kg-1),DLX(DLX 20 mg·kg-1+reserpine 5 mg·kg-1)and ZBH2012001(ZBH2012001 5,10 and 20 mg·kg-1+reserpine 5 mg·kg-1)groups.One hour following drug administration,reserpine was injected intraperitoneally to establish a monoamine-depletion model.The ptosis,akinesia,and hypothermia assays were performed to evaluate the effect of ZBH2012001 on the down-regulation of the reserpine-induced monoamine system.The TST in mice was used to evaluate the effect of ZBH2012001 on reserpine-induced depressive-like behavior while high-performance liquid chromatography with electrochemical detection(HPLC-ECD)was used to measure the levels of monoamines and their metabolites in the hippocampal tissue of reserpine-induced monoamine-depletion mice.ELISA was employed to detect the contents of tumor necrosis factor-alpha(TNF-α)and interleukin-6(IL-6)in the hippocampal tissue of reserpine-induced monoamine-depletion mice.Western blotting was used to assess the expressions of ionized calcium-binding adapter molecule-1(Iba-1)and nuclear factor-kappa B(NF-κB)in the hippocampal tissue of reserpine-induced monoamine-depletion mice.RESULTS ① Compared with the vehicle group,ZBH2012001(5,10 and 20 mg·kg-1)significantly reduced the immobility time both in the TST in mice(P<0.01,respectively),and ZBH2012001(20 mg·kg-1)and in the FST in mice(P<0.05).② ZBH2012001 competitively inhibited the binding of[3H]-imipramine to hSERTs and[3H]-nisoxetine to hNETs,with the half maximal inhibitory concentration(IC50)values of 84.95 and 712.90 nmol·L-1,respectively.③Com-pared with the vehicle group,ZBH2012001(10 and 20 mg·kg-1)significantly increased the head twitches induced by 5-HTP in mice(P<0.01,respectively)and increased the mortality rate in mice induced by yohimbine(P<0.05,P<0.01).④ In the reserpine-induced monoamine-depletion model in mice,compared with the vehicle group,mice in the reserpine model group exhibited ptosis,akinesia and hypothermia feature(P<0.01,respectively),significantly prolonged immobility time in the TST(P<0.01),significantly decreased the levels of NE,5-HT and dopamine(DA)(P<0.05,P<0.01),significantly increased the metabolic conversion rate of 5-HT and DA(P<0.01,respectively),significantly elevated levels of TNF-α and IL-6(P<0.05,respectively),and significantly increased expressions of Iba-1 and NF-κB(P<0.05,respectively)in the hippocampus.Compared with the model group,ZBH2012001(5,10 and 20 mg·kg-1)significantly antagonized ptosis and hypothermia behaviors induced by reserpine(P<0.01,respectively),ZBH2012001(10 and 20 mg·kg-1)significantly shortened the immobility time in reserpine-treated mice(P<0.05,P<0.01),ZBH2012001(20 mg·kg-1)significantly increased the levels of NE and 5-HT in the hippocampus of reserpine-treated mice(P<0.05,respectively),decreased the metabolic conversion rate of 5-HT(P<0.05),significantly reduced the contents of TNF-α and IL-6 in the hippocampus of reserpine-treated mice(P<0.05,respectively),ZBH2012001(5,10 and 20 mg·kg-1)significantly reduced the expression of Iba-1 protein in the hippocampus of reserpine-treated mice(P<0.01,respec-tively),and ZBH2012001(20 mg·kg-1)significantly reduced the expression of NF-κB protein in the hippocampus of reserpine-treated mice(P<0.05).CONCLUSION ZBH2012001 exerts its antidepres-sant effect through a dual mechanism involving monoamine enhancement and inflammation suppres-sion.

ObjectiveThrough improving the potential of vascular endothelial growth factor receptor (VEGFR)-humanized mouse model (hKDR^+/+) with C57BL/6N background to allow the growth of different mouse tumor cell lines, to establish novel tumor-bearing mouse models which can support in vivo tumorigenesis of different mouse tumor cell lines and be used to evaluate drugs targeting VEGFR.MethodsFirstly, a method to evaluate the in vivo activity of antibody targeting VEGFR based on the hKDR^+/+ humanized mouse model was established. Recombinant activating gene 1 (Rag1) knockout mice (Rag1^-/-) were established using CRISPR/Cas9 technology. Then these Rag1^-/- mice were crossed with hKDR^+/+ mice to get a double gene modified homozygous hKDR^+/+/Rag1^-/- mouse model by screening. Finally, tumor cell lines derived from different mouse strains were tested on the double gene-modified mouse model to compare the differences in tumor growth. ResultsAntibodies designed for VEGFR showed significant anti-tumor activity in hKDR^+/+ mice, which significantly reduced tumor volume and weight compared with the PBS group (P<0.01, P<0.05). The number of B cells and T cells in the peripheral blood of Rag1^-/- mice and hKDR^+/+/Rag1^-/- mice decreased (P<0.05, P<0.001). Tumors were observed in hKDR^+/+/Rag1^-/-, Rag1^-/-, wild-type, and hKDR^+/+ mice after 7 d of inoculation of MC38 cells derived from C57BL/6 mice. Tumors were only observed in groups of hKDR^+/+/Rag1^-/- and Rag1^-/- mice, but not in the wild-type and hKDR^+/+ mice after 10 d of inoculation with CT26 cells derived from BALB/c mice. After 3 weeks of inoculation, the tumor volume of hKDR^+/+/Rag1^-/- mice was significantly larger than that of Rag1^-/- mice (P<0.01). ConclusionRag1 knockout mice were obtained and a novel hKDR^+/+/Rag1^-/- double genes modified mouse model was further screened. The tumor cell lines from different mouse strain origins were more prone to growth in mice with Rag1 gene deficiency. The results suggest that the reduced immune response of hKDR^+/+ humanized mice will improve the capacity of supporting the growth of mouse tumor lines in the model. As a result, more tumor-bearing mouse models may be constructed for the evaluation of drugs targeting VEGFR in this way.

Objective: To explore the effect and mechanism of omega 3-polyunsaturated fatty acid(ω3-PUFA) dietary intervention on mitochondrial function of white adipose tissue in adult rats with postnatal early overfeeding. Methods: An overfed animal model by adjusting litter size was developed for the study of neonatal overfeeding. The litter size was adjusted to 3 male rats per litter(small litter, SL group) and 10 pups per litter(normal litter, NL group). After weaning(week 3), the pups were fed standard chow or ω3-PUFA diet(SL-FO) until postnatal weeks 13. Food intake, body weight, and rectal temperature of rats were measured regularly, and energy metabolism of animals was monitored in week 13. During week 3 and 13, subcutaneous adipose tissue was collected. Inguinal preadipocytes of mice were isolated and induced to differentiate, and 50 μmol/L eicosapentaenoicacid(EPA) was administered for 48 h at the late stage of differentiation. The mRNA and protein expression levels of mitochondrial related genes, mitochondrial copy number, and oxygen consumption rate of adipocytes were detected in adipose tissue and adipocytes. Results: By the 3rd week, the body weight, food intake, and fat cell area in SL group were higher than those in NL group while the body temperature was lower until to 13 weeks. By the 13th week, the O₂ consumption, CO₂ output, and heat production of rats in SL group were lower than those in NL group. Meanwhile, the expressions of mitochondrial function related genes such as uncoupling protein 1(UCP1), carnitine palmitoyltransferase 1(CPT1), SIRT1, and mitochondrial biosynthesis regulatory gene peroxisome proliferator-activated receptor coativator-1 (PGC1α) in adipose tissue by the 3rd and 13th week were significantly reduced(P<0.05). After weaning, ω3-PUFA diet significantly reduced weight gain in SL rats, increased UCP1 protein expression, restored energy metabolism level and mitochondrial function related gene expression. In vitro intervention of EPA increased the mitochondrial copy number, the mRNA and protein expression levels of mitochondrial biosynthesis and functional genes, as well as the mitochondrial basic oxygen consumption rate(P<0.05). Conclusion ω3-PUFA improves postnatal overfeeding-induced impairment of the mitochondrial function and biosynthesis of subcutaneous white adipose tissue in rats, which may be an important mechanism for fish oil diet to inhibit the early over-nutrition program and restore the thermogenic metabolism.

Objective To obtain the basic growth parameters of a self-established F1 hybrid CB6F1 C57-ras transgenic mouse model, and to provide basic information for commercialization of this mouse model. Methods F1 hybrid mice (CB6F1) were produced by crossing C57-ras heterozygous transgenic (c-Ha-ras+/-) male mice and wild-type BALB/cJ female mice.The average litter size, weaning rate, sex ratio, growth performance and C57-ras transgenic positive rate were recorded and analyzed.Results The average litter size was eight, weaning rate was 90%, and sex ratio was approximately in accordance with prediction.The average body weight of newborn mice was 1.73 ±0.05 g.The average body weight of 10-week old c-Ha-ras transgenic female and male mice in CB6F1 background was 24.38 ±1.74 g and 29.42 ±1.72g, respectively, which had a significant difference (P<0.01).The c-Ha-ras transgenic positive rate was 46.9%. which was in accordance with genetic rules.Conclusions The F1 hybrid mice (CB6F1) produced by crossing C57-ras heterozygous transgenic ( c-Ha-ras +/-) male mice and wild-type BALB/cJ female mice show normal growth performance and development characteristics, and it can be used for large-scale commercial supply.

Objective To determine the normal range of hematological and visceral weight parameters of F 1 4-week and 8-week old, male and female transgenic CB6F1 mice.The influence of gender and week age on the biochemical parameters was assessed .Methods 4-week and 8-week old CB6F1 mice, half male and half female ( n=20 in each group) , were weighed alive , dissected to weigh the main viscera , and blood samples were collected to test the physiological and biochemical parameters .Results When 4-week old and 8-week old CB6F1 mice were compared , there were significant differences in 22 parameters (body weight, heart, liver, spleen, left ovary, left testis, right testis, WBC, RBC, HGB, HCT, MCV, MCH, PCT, MPV, PDW, LYM, TP, ALT, ALB, P and TG) (P<0.01 for all), and in 8 parameters (left kidney, right kidney, right adrenal, thymus, left ovary, RDW, MON%and BUN) (P<0.05 for all). When male and female 4-week CB6F1 mice were compared, there were significant differences in 14 parameters ( body weight, heart, liver, spleen, lung, left kidney, right kidney, MCHC, LYM, ALT, ALP, GLU, P and CHO) (P<0.01 for all), and in 6 parameters (right adrenal, WBC, PCT, MPV, TP and BUN) (P<0.05 for all).For male and female 8-week old CB6F1 mice, there were significant differences in 15 parameters (body weight, heart, liver, lung, left kidney, right kidney, MCV, PCT, LYM, LYM%, NEUT%, ALT, GLU, P and CHO) (P<0.01 for all), and in 5 parameters (WBC, RBC, MPV, NEUT and TP) (P<0.05 for all).Conclusions The normal range of hematological and visceral weight parameters of 4-week and 8-week old male and female CB6F1 mice are determined.Our study establishes normal detection indexes of CB6F1 mice and provides useful reference for its application .

Objective To improve the gene targeting efficiency with C57BL/6 embryonic stem ( ES) cells.Meth-ods Three different genetically modified C57BL/6 ES cell lines, named TLX3, Ai3K and SL, were microinjected into ICR, B6( Cg)-Tyrc-2J and BALB/c mouse blastocysts, respectively.The efficiency was statistically evaluated according to three aspects:blastocyst collection, chimera production and germline transmission.Results None of the three ES cell lines was germline transmitted with B6(Cg)-Tyrc-2J mice as blastocyst donors, while it was achieved with both BALB/c and ICR mouse blastocysts.Compared in the aspect of blastocysts collection, ICR mouse was much better than BALB/c mouse (P<0.05), and the chimera production efficiency of ICR mouse was comparable to that of BALB/c mouse (P =0.115). As to the germline transmission efficiency, that of BALB/c mice is significantly higher than that of the ICR mice ( P<0.01).Conclusions The germline transmission efficiency of BALB/c mouse is highest among these three mouse strains. However, it has the disadvantages of blastocyst collection, developmental delay and zona pellucida fragility, compared with ICR mouse.Therefore, ICR mouse is also a good candidate as blastocyst donor for embryonic stem cell microinjection.

Objective To establish a simple , fast and economic total DNA extraction method to serve the rapid identification of model mouse genotype in large number of mice .Methods Three methods, i.e.phenol extraction, isopropyl alcohol precipitation and mouse ear boiling methods were used to extract the total DNA from ten C 57-rasmodel mice.The purity and yield of DNA obtained by the three methods were compared , and polymerase chain reaction ( PCR) assay was used to compare the efficacy of the three extraction methods .Results Among the three methods , phenol extraction was the best and isopropyl alcohol precipitation was the poorest in DNA yield .In terms of DNA purity , the phenol extraction was the best and the mouse ear boiling method was the poorest .All the three methods could be used to extract the total DNA from mice serving as template of PCR reaction for the mouse genotype identification .The time consumption of three methods are 12.5 hr ,13 hr and 0.18 hr.Mouse ear boiling method was significantly lower than that of the other two methods ( P <0.01 ) ,.The obtained total DNA can be stored at conventional -20℃for 7 days and 30 days later still can be used as a template for PCR reaction .Conclusions Among the three methods studied , the mouse ear boiling method is simple and with the lowest cost , so it is feasible for total DNA extraction in scaled genotyping experiments .

OBJECTIVETo construct a three-dimensional (3D) model of arteries supplying the extrahepatic bile duct with a new segmentation algorithm based on submillimeter CT data.

METHODSThe new image segmentation algorithm based on interactive volume rendering was integrated into Medical Image Three-Dimensional Visualization System (MI-3DVS) as an intersected plug-in. The abdominal submillimeter CTA data of 10 patients were imported into MI-3DVS and the 3D model of the extrahepatic bile duct and its supplying arteries were constructed. The 3D model was zoomed in, zoomed out and spinned for observation and analysis of the arteries supplying the extrahepatic bile duct.

RESULTSThe 3D models of the blood supply to extrahepatic bile duct allowed stereoscopic, and accurate display of the fourth- and fifth-level branches of the hepatic artery, the second-level branches of the cystic artery, the pancreatic duodenal artery arch and the retroportal artery. The 3D models also provided a clear vision of the biliary structures including the hepatobiliary tract, the left and right hepatic ducts, gallbladder, the liver duct, and the common bile duct.

CONCLUSIONBased on the segmentation method of interactive volume rendering, the CT data of the arterioles supplying the extrahepatic bile duct can be extracted and segmented for 3D reconstruction to display the three-dimensional anatomical structures of the extrahepatic bile duct and its supplying arteries.