OBJECTIVE: To construct a modABC gene knockout strain of Proteus mirabilis and explore the effect of modABC gene deletion on biological characteristics of Proteus mirabilis. METHODS: Fusion PCR was used to obtain the fusion gene of modABC and the kanamycin-resistant gene Kn, which was ligated with the suicide vector pCVD442 and transduced into Proteus mirabilis. The modABC gene knockout strain of Proteus mirabilis was obtained after homologous recombination with the suicide vector. PCR and Sanger sequencing were used to identify genomic deletion of modABC gene in the genetically modified strain. The concentration of molybdate in the wild-type and gene knockout strains was determined using inductively coupled plasma mass spectrometry (ICP-MS), and their survival ability in LB medium was compared under both aerobic and anaerobic conditions. RESULTS: PCR and sanger sequencing confirmed genomic deletion of modABC gene in the obtained Proteus mirabilis strain. The concentration of intracellular molybdenum in the modABC gene knockout strain was 1.22 mg/kg, significantly lower than that in the wild-type strain (1.46 mg/kg, P < 0.001). Under the aerobic condition, the modABC gene knockout strain grown in LB medium showed no significant changes in survival ability compared with the wild-type strain, but its proliferation rate decreased significantly under the anaerobic condition and also when cultured in nitrate-containing LB medium under anaerobic condition. CONCLUSION Homologous recombination with the suicide vector can be used for modABC gene knockout in Proteus mirabilis. modABC gene participates in molybdate uptake and is associated with anaerobic growth of Proteus mirabilis in the presence of nitrate.
Humans ; Gene Deletion ; Nitrates ; Proteus mirabilis/genetics* ; Gene Knockout Techniques

Establishing a suitable animal model is important for studying the mechanism of inflammatory bowel disease (IBD) and exploring new therapeutic approaches. Although a large number of IBD single gene knockout animal models have been established, single knockout of certain genes associated with human IBD susceptibility does not manifest symptoms of IBD or manifest extremely milder symptoms, while composite animal models based on other modeling factors can better simulate the clinical features of IBD. This article mainly introduces three novel composite animal models and elaborates the possible pathogenesis of each composite model:animal models established by gene double knockout have more obvious and earlier symptoms than single-knockout models; single gene knockout model with Helicobacter infection can help to study the role of microbial infections in the pathogenesis of IBD; on the basis of gene knockout, specific deletion of certain immune cells can be used to study the role of the immune cells in the development of IBD. Among the above composite animal models, double knockout mice may be important animal models for IBD study.
Animals ; Disease Models, Animal ; Gene Knockout Techniques ; Humans ; Inflammatory Bowel Diseases ; genetics ; immunology ; Mice, Knockout ; Research

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.
Animals ; Mice ; Mice, Knockout ; CRISPR-Cas Systems ; Blood Pressure ; Gene Knockout Techniques ; Zygote

The effective material basis of traditional Chinese medicine(TCM) is an indispensable part of studies on TCM, and each technology has its advantages and disadvantages. The target constituent knock-out/knock-in technology has attracted much attention since it was proposed because of its unique advantages of regarding the extract of the formula as a whole, which can better reflect the characteristics of multi-component and multi-target integration and regulation of TCM. This method investigated the contribution of target constituent to the overall efficacy of a TCM by analyzing the changes in efficacy of the remaining formula before and after knock-out/knock-in of the target constitution. The application of this model not only facilitates studies of the effective constituents of TCM, but also help to develop the quality control standard of TCM. However, the application of this model is restricted due to the limitation of target constituent separation technology. By reviewing the literatures in recent years, this study summarized the research process and application of this method for a reference.
Animals ; Drugs, Chinese Herbal/pharmacology* ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Medicine, Chinese Traditional ; Quality Control

The establishment and development of gene knockout mice have provided powerful support for the study of gene function and the treatment of human diseases. Gene targeting and gene trap are two techniques for generating gene knockout mice from embryonic stem cells. Gene targeting replaces endogenous knockout gene by homologous recombination. There are two ways to knock out target genes: promoter trap and polyA trap. In recent years, many new gene knockout techniques have been developed, including Cre/loxP system, CRISP/Cas9 system, latest ZFN technology and TALEN technology. This article focuses on the several new knockout mouse techniques.
Animals ; Disease Models, Animal ; Embryonic Stem Cells ; Gene Knockout Techniques ; trends ; Gene Targeting ; trends ; Homologous Recombination ; Humans ; Mice ; Mice, Knockout

The aim of the study was to establish Ace2 (angiotensin-converting enzyme 2) knockout mouse model with CRISPR/Cas9 gene targeting technology. A vector targeting Ace2 gene knockout was constructed with the primers of single-guide RNA (gRNA), and then transcribed gRNA/Cas9 mRNA was micro-injected into the mouse zygote. The deletion of exons 3 to 18 of Ace2 gene in mice was detected and identified by PCR and gene sequencing. The Ace2 gene knock-out mice were bred and copulated. Ace2 protein and mRNA expression were detected by Western blot and qRT-PCR in F3 progeny knock-out male mice. The gRNA expression vector was successfully constructed and transcribed in vitro, and active gRNA and Cas9 mRNA were injected directly into zygote. The deletion of exons 3 to 18 of Ace2 gene in six positive founder mice as the F0 generation were confirmed by PCR and gene sequencing. Six founder mice were mated with wild-type mice, then achieved F1 generation were mated and produced F2 generation. The female positive mouse of F2 was selected to mate with wild-type mice and produce Ace2 mice of F3 generation. Ace2 mRNA and protein were not detected in tissues of these Ace2 mice. In conclusion, a mouse model with Ace2 deficiency has been successfully established with CRISPR/Cas9 technique, which shall lay a foundation for future investigation of Ace2.
Animals ; CRISPR-Cas Systems ; Female ; Gene Knockout Techniques ; Gene Targeting ; Male ; Mice ; Mice, Knockout ; RNA, Guide ; genetics

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.
Animals ; Animals, Genetically Modified ; Gene Knockout Techniques ; HLA Antigens ; Humans ; Nuclear Transfer Techniques ; Swine ; Transplantation, Heterologous

To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1, we reported the construction and functional research of a stable TrxR1 gene (encode thioredoxin reductase 1) knockout HCT-116 cell line. We designed and selected TrxR1 knockout sites according to the TrxR1 gene sequence and CRISPR/Cas9 target designing principles. SgRNA oligos based on the selected TrxR1 knockout sites were obtained. Next, we constructed knockout plasmid by cloning the sgRNA into the pCasCMV-Puro-U6 vector. After transfection of the plasmid into HCT-116 cells, TrxR1 knockout HCT-116 cells were selected using puromycin resistance. The TrxR1 knockout efficiency was identified and verified by DNA sequencing, immunoblotting, TRFS-green fluorescent probe, and cellular TrxR1 enzyme activity detection. Finally, the correlation between TrxR1 expression and cellular effects of drugs specifically targeting TrxR1 was investigated by CCK-8 assay. The results demonstrated that the knockout plasmid expressing the sgRNA effectively knocked-out TrxR1 gene within HCT-116 cells, and no expression of TrxR1 protein could be observed in stable TrxR1 knockout HCT-116 (HCT116-TrxR1-KO) cells. The TrxR1-targeting inhibitor auranofin did not show any inhibitory activity against either cellular TrxR1 enzyme activity or cell proliferation. Based on these results, we conclude that a stable TrxR1 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques, which may facilitate investigating the role of TrxR1 in various diseases.
CRISPR-Cas Systems/genetics* ; Gene Editing ; Gene Knockout Techniques ; HCT116 Cells ; Humans ; RNA, Guide/metabolism*

OBJECTIVETo breed estrogen receptor beta (ERbeta) gene knock-out female mice for studying postmenopausal osteoporotic fracture.

METHODSThree pairs of ERbeta gene knock-out mice were bred for 3 months, and 14 2-month-old female wild-type C57BL/6J mice with the same genetic background were paired at the ratio of 2:1 and mated with the male ERbeta gene knock-out homozygote mice. After further breeding to obtain sufficient number of mice, the genome DNA was extracted from the tail of the mice for genotyping by PCR. Ten 4-month-old female filial mice with ERbeta gene knock-out and 10 wild-type female mice were randomly selected and sacrificed, and the right proximal tibiae were removed and subjected to micro CT for measuring the parameters of trabecular bone histomorphometry.

RESULTSA total of 340 filial generation mice were reproduced in 2 months and genotypic identification revealed a proportion of ERbeta+ or + mice of 23.5%, ERbeta+ or - mice of 48.27 percent; and homozygous mutant (ERbeta- or -) mice of 28.3% (in which 54 were female). The MicroCT data revealed that the micro-architecture of the proximal tibiae was significantly different between ERbeta gene knock-out mice selected from the filial generation and wild type mice (P<0.05).

CONCLUSIONIt is feasible to breed ERbeta knock-out female mice by introducing female wild-type mice to pair and mate with ERbeta knock-out homozygote male mice. This approach allows breeding of sufficient number of female ERbeta knock-out mice as the animal models for studying the role of ERbeta.

Animals ; Breeding ; DNA ; analysis ; Estrogen Receptor beta ; genetics ; Female ; Gene Knockout Techniques ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout

OBJECTIVE: To investigate the effects of CACNA1H gene knockout (KO) on autistic-like behaviors and the morphology of hippocampal neurons in mice. METHODS: In the study, 25 CACNA1H KO mice of 3-4 weeks old and C57BL/6 background were recruited as the experimental group, and 26 wild type (WT) mice of the same age and background were recruited as the control group. Three-chamber test and open field test were used to observe the social interaction, anxiety, and repetitive behaviors in mice. After that, their brain weight and size were measured, and the number of hippocampal neurons were observed by Nissl staining. Furthermore, the CACNA1H heterozygote mice were interbred with Thy1-GFP-O mice to generate CACNA1H^-/--Thy1⁺(KO-GFP) and CACNA1H^+/+-Thy1⁺ (WT-GFP) mice. The density and maturity of dendritic spines of hippocampal neurons were observed. RESULTS: In the sociability test session of the three-chamber test, the KO mice spent more time in the chamber of the stranger mice than in the object one (F_{1, 14}=95.086, P < 0.05; Post-Hoc: P < 0.05), without any significant difference for the explored preference index between the two groups (t=1.044, P>0.05). However, in the social novelty recognition test session, no difference was observed between the time of the KO mice spend in the chamber of new stranger mice and the stranger one (F_{1, 14}=18.062, P < 0.05; Post-Hoc: P>0.05), and the explored preference index of the KO mice was less than that of the control group (t=2.390, P < 0.05). In the open field test, the KO mice spent less time in the center of the open field apparatus than the control group (t=2.503, P < 0.05), but the self-grooming time was significantly increased compared with the control group (t=-2.299, P < 0.05). Morphological results showed that the brain weight/body weight ratio (t=0.356, P>0.05) and brain size (t=-0.660, P>0.05) of the KO mice were not significantly different from those of the control group, but the number of neurons were significantly reduced in hippocampal dentate gyrus compared with the control group (t=2.323, P < 0.05). Moreover, the density of dendritic spine of dentate gyrus neurons in the KO-GFP mice was significantly increased compared with the control group (t=-2.374, P < 0.05), without any significant difference in spine maturity (t=-1.935, P>0.05). CONCLUSION CACNA1H KO mice represent autistic-like behavior, which may be related to the decrease in the number of neurons and the increase in the density of dendritic spine in the dentate gyrus.
Animals ; Autistic Disorder/genetics* ; Calcium Channels, T-Type/genetics* ; Gene Knockout Techniques ; Hippocampus ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Neurons