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.

The luciferase reporter system of the MMP12 endogenous promoter for investigating transcriptional regulation of the human MMP12 gene

Background: Matrix metalloproteinase 12 (MMP12), a member of MMPs, can take lots of roles including extracellular matrix component degradation, viral infection, inflammation, tissue remodeling and tumorigenesis. To explore the transcriptional regulation of MMP12 gene, a sensitive luciferase reporter HEK293 cell line for endogenous MMP12 promoter was generated by CRISPR/Cas9 technology. Results: The HEK293-MMP12-T2A-luciferase-KI cell line was successfully established by CRISPR/Cas9 technology. The sequencing results indicated that one allele of the genome was proven to have a site-directed insertion of luciferase gene and another allele of the genome was confirmed to have additional 48 bp insertion in this cell line. The cell line was further demonstrated to be a sensitive reporter of the endogenous MMP12 promoter by applying transcription factors STAT3, AP-1 and SP-1 to the cell line. The reporter cell line was then screened with bioactive small molecule library, and a small molecule Tanshinone I was found to significantly inhibit the transcriptional activity of MMP12 gene in HEK293-MMP12-T2A-luciferase-KI cell line by luciferase activity assay, which was further confirmed to inhibit the expression of MMP12 mRNA in wild-type HEK293 cells. Conclusions: This novel luciferase knock-in reporter system will be helpful for investigating the transcriptional regulation of MMP12 gene and screening the drugs targeting MMP12 gene.

Development and verification of an FLP/FRT system for gene editing in Bacillus licheniformis / 生物工程学报

Few tools of gene editing have been developed in Bacillus licheniformis at present. In order to enrich the tools, an FLP/FRT gene editing system that can repeatedly use a single selectable marker was constructed in Bacillus licheniformis, and the system was verified by knocking out an alpha amylase gene (amyL), an protease gene (aprE) and knocking in an exogenous Vitreoscilla hemoglobin gene (vgb). First, knock-out plasmids pNZTT-AFKF of amyL and pNZTT-EFKF of aprE were constructed using thermosensitive plasmid pNZT1 as a carrier. The two knock-out plasmids contained respective homology arms, resistance genes and FRT sites. Then the knock-out plasmids were transformed into Bacillus licheniformis and the target genes were replaced by respective deletion cassette via twice homologous exchange. Finally, an expression plasmid containing FLP recombinase reading frane was introduced and mediated the excision of resistance marker. In order to expand the practicability of the system, knock-in plasmid pNZTK-PFTF-vgb was constructed, with which knock-in of vgb at pflB site was carried out successfully. The results showed that amyL and aprE were successfully knocked out and the marker kanamycin cassette exactly excised. The activities of amylase and protease of deletion mutants were reduced by 95.3% and 80.4% respectively. vgb was successfully knocked in at pflB site and the marker tetracycline cassette excised. The expression of integrated vgb was verified via real-time PCR. It is the first time to construct an FLP/FRT system for gene editing in Bacillus licheniformis, which could provide an effective technical means for genetic modification.

TMEM43-S358L mutation enhances NF-κB-TGFβ signal cascade in arrhythmogenic right ventricular dysplasia/cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is a genetic cardiac muscle disease that accounts for approximately 30% sudden cardiac death in young adults. The Ser358Leu mutation of transmembrane protein 43 (TMEM43) was commonly identified in the patients of highly lethal and fully penetrant ARVD subtype, ARVD5. Here, we generated TMEM43 S358L mouse to explore the underlying mechanism. This mouse strain showed the classic pathologies of ARVD patients, including structural abnormalities and cardiac fibrofatty. TMEM43 S358L mutation led to hyper-activated nuclear factor κB (NF-κB) activation in heart tissues and primary cardiomyocyte cells. Importantly, this hyper activation of NF-κB directly drove the expression of pro-fibrotic gene, transforming growth factor beta (TGFβ1), and enhanced downstream signal, indicating that TMEM43 S358L mutation up-regulates NF-κB-TGFβ signal cascade during ARVD cardiac fibrosis. Our study partially reveals the regulatory mechanism of ARVD development.

Advances in targeted replacement genome editing in plants / 生物工程学报

Targeted replacement genome editing refers to DNA modification and engineering technology that could induce and achieve mutations of targeted gene replacement or knockin at a target gene or DNA region. In this review, the principles, implementation methods, factors that influence efficiency and accuracy, and applications of gene replacement editing were summarized and discussed. It provides the reference for gene functional characterization and genetic improvements through gene replacement strategies in higher plant especially crops.

Evaluation of the CaV1 1-R528 H gene knock-in mouse model of thyrotoxic hypokalemic periodic paralysis / 中国实验动物学报

Objective To establish and evaluate the CaV1?1?R528H gene knock?in mouse model of thyrotoxic hy?pokalemic periodic paralysis. Methods Thirty?six 8?week?old male CaV1?1?R528H gene knock?in mice and thirty?six 8?week?old wild?type male C57BL/6J mice were used in this study. Using three?factor two?level 2 × 2 × 2 factorial design ( the three factors including mutation, thyroxine and insulin, and two levels were with or without) , the mice were divided into 8 groups. The thyroxine groups were intraperitoneally injected with levothyroxine in a dose of 350 μg/kg once per day for 12 consecutive days to produce thyrotoxicosis. The insulin groups were intraperitoneally injected with short?acting insulin in a dose of 0?8 U/kg after the last administration of levothyroxine, and the potassium levels of different groups were meas?ured and recorded before (0 min) and after insulin injection (30 min, 60 min). Results (1) Compared with the control group, the following phenomena including irritability, dull coat, increased diet and water intake, and slow body weight gain, were observed in the thyrotoxic mice. Thyroid function tests showed that the levels of T3 and T4 in the thyrotoxic mice were significantly higher than those in the corresponding control mice (P<0?05), and the TSH level was significantly low?er than that of the corresponding control mice (P<0?05 ). (2) After administration of insulin or thyroxine alone, the po?tassium levels in the mutant and wild?type mice were not significantly different. However, after combined administration of thyroxine and insulin, the potassium levels in the mutant group were significantly lower than those in the wild?type mice at 30 min and 60 min ( P<0?05 for both). (3) The main effects and interactions:Mutation factor or thyroxine factor alone did not influence on the potassium level, only insulin showed hypokalemic effect (P<0?05). There were interactions be?tween thyroxine and mutation, and between insulin and mutation (P<0?05), but no interaction between thyroxine and in?sulin. Conclusions (1) A thyrotoxicosis state in mice is successfully developed in this study. (2) An CaV1?1?R528H gene knock?in mouse model of thyrotoxic hypokalemic periodic paralysis is successfully established.

Research progress on effective compounds group of Chinese materia medica / 中草药

Effective compounds group of Chinese materia medica (CMM) is a pharmacological active compounds group containing all components that are closely related to their clinical application. It plays a core role in CMM formula. We think it maintains not only the characteristics of the whole system and the system integrity of CMM formula, but also their chemical compositions are clear. However, due to the complexity of CMM, the successful research cases are very few. In this paper, we summarized several outstanding research cases of effective compounds group in recent years and there are three strategies, including "research on decomposed-combined recipes of CMM based on assessment of chemical components in CMM way", "screening the effective components group based on knock-in and knock-out from chromatographic fingerprint", and "screening the effective components group based on serum pharmacochemistry". We also summarized the ways to illustrate properties of effective compounds group, such as combination of multiple component, clear chemical composition, and system integrity. This paper provided ideas for the future research of effective components of CMM.

Analysis on ideas and methods to identify target components group of Chinese materia medica preparation / 中草药

The preparation of Chinese materia medica (CMM) possesses the characteristic of holistic function involving multi-component, multi-target, and multi-link. This characters make it difficult to optimize the preparation process and construct the quality standards of CMM. Thus process building and quality evaluation system based on correctly identifying the components that closely related to the efficacy is a key scientific issue in the preparation process design and quality control of CMM. In this paper, a more comprehensive analysis on the feasibility of different research methods was carried out, including the spectral-efficiency relationships, pharmacokinetics, serum medicinal chemistry and serum pharmacology of drugs, the effect of integration effect correlation analysis, virtual screening, and comparative analysis of knock-in/knock-out. The purpose of this article is to provide the reference for identifying the bioactive marker group which is used for the CMM preparation process and quality evaluation.

Construction of Cchl1 a3 gene R528 H knock-in mouse model related to hypokalemic periodic paralysis / 中国比较医学杂志

Objective To construct Cchl1a3 gene R528H knock-in mouse model related to hypokalemic periodic paralysis.Methods ES cells were transfected with Cchl1a3-Konckin targeting vector linearized by Not I digestion , selected in the medium containing both G 418 and ganciclovoir .Resistant clones were screened by PCR and further confirmed by DNA sequencing for correct homologous recombinants .Chimera mice were obtained by routing microinjection of homologous recombined ES cells into blastocysts .Heterozygous mice were obtained by mating .Through heterozygous mice with FLP mice mating , removal of neo gene heterozygous mice were established and identified with the PCR and DNA sequencing . After mating, homozygous offspring were constructed and observed .Results ES cells were successfully transfected withtargeting vector .It was confirmed that 9 resistant clones happened right homologous recombination by PCR and DNA sequencing .7 chimera mice were obtained by microinjection .After breeding the chimeric mice , heterozygous mice were mated FLP mice to obtain 9 heterozygous mice removal of neo gene, the finally obtained 15 homozygous mice with Flp-deleted neo gene.In the developmental stage of sexual maturity , the spirit of the mice, restaurants and activities in good condition, but the gradual emergence of hair removal at 4 months of age, skin ulceration and even death .Conclusions We successfully constructed Cchl1a3 gene R528H mutation homozygous mice.And it laid a foundation for the study of human CACNA1S gene function and to clarify the molecular mechanism of hypokalemic periodic paralysis .

Generation of Adiponectin Gene Knock-out and LacZ Gene Knock-in Mouse Model / 生物化学与生物物理进展

Adiponectin is an adipocyte-derived secretory protein. It was found to be associated with insulin resistance, inflammation and arteriosclerosis. To further study the biological function and expression of adiponection in vivo, adipoenctin gene knock-out and LacZ gene knock-in mouse model was constructed. Gene targeting strategy was designed to replace part of exon 2 and exon 3 of adiponectin gene with full length LacZ gene in frame with remaining upstream ATG and signal peptide sequence of exon 2. The targeting vector (Adipo-LacZ-XpPNT) was constructed and verified by restriction enzyme digestion and sequencing. CJ7 ES cells were transfected with targeting vector linearized by NotⅠ digestion, selected in the medium containing both G418 and ganciclovoir. Resistant clones were screened by PCR and further confirmed by Southern blot for correct homologous recombinants. Chimera mice were obtained by routing microinjection of homologous recombined ES cells into blastocysts. After mating, mice heterozygous and further homozygous for adiponectin knockout and LacZ gene knock-in were established. Expression of both endogenous adiponectin and exogenous LacZ gene in mouse tissues and sera were detected by RT-PCR, Northern-blot, Western blot and ELISA. The results show that adiponectin was disrupted at both mRNA and protein levels. LacZ gene is expressed exclusively in adipose tissue of mutant mice. Its expression profile is identical to endogenous adiponection. Unexpectedly, LacZ activity could not be detected in both adipose tissue and serum although LacZ protein can be detected in adipose tissue but not in serum of mutant mice. In conclusion, mice homozygous for adiponectin knockout and LacZ gene knock-in have been successfully constructed. Mutant mice display LacZ expression profile identical to endogenous adiponectin albeit neither LacZ activity nor protein can be detected in serum of mutant mice.

Novel patterns of efficient components recognition and quality control for Chinese materia medica based on constituent knock-out/knock-in / 中草药

The patterns of efficient components recognition and quality control for Chinese materia medica (CMM) have been the difficult and hot topics for CMM modernization.To get a radical and significant breakthrough in the investigation on the efficient component recognition and quality control standard for CMM,a tentative idea about efficient component recognition and quality control pattern for CMM based on constituent knock-out/knock-in is initially proposed in this article on the foundation of retrospective and prospective analyses.And its main aim is to provide some creative and practical ideas and methods to recognize the key efficient components of CMM precisely and quickly,and to meet with the requirements that the quality control standards for CMM will be effectiveness-related,quantitative and accurate,controllable and assessable.

DISTRIBUTION OF GFP-POSITIVE NEURONS AND THE COLOCALIZATION WITH PARVALBUMIN IN THE CAUDAL SPINAL TRIGEMINAL NUCLEUS IN THE GAD67-GFP KNOCK-IN MOUSE / 解剖学报

Objective To observe the distribution of GABA-containing neurons revealed by GFP expression and the colocalization of the GFP with parvalbumin(PV) in the caudal spinal trigeminal nucleus(Vc),the glutamate decarboxylase 67-green fluorescence protein(GAD67-GFP) knock-in mice were used in the present study. Methods Double-labeled techniques were used by in situ hybridization combined with immunohistochemistry for GFP and double immunofluorescence histochemistry for GFP and NeuN(neuronal nuclei protein,a neuronal marker) or PV.The stained sections were observed under light microscope and a confocal laser-scanning microscope. Results 1.Over 90% of GFP-positive neuronal cell bodies in the laminae Ⅰ and Ⅱ of the Vc showed hybridization signals for GAD67 mRNA,and that almost all neuronal cell bodies with GAD67 mRNA signals were GFP-positive;2.GFP-positive neurons were mainly distributed in the laminae Ⅰ and Ⅱ of the Vc and the vast majority of them were small neurons.The considerable number of GFP-positive processes and somata were most densely observed in the lamina Ⅱ of the Vc.The proportion of GFP-positive neurons in the NeuN-labeled neurons of the Vc was about 19.4% and 24.3% in laminae Ⅰ and Ⅱ,respectively;3.Double-labeled neurons for GFP/PV are mainly found in laminae Ⅰ and Ⅱ of the Vc.The proportion of GFP/PV double-labeled neurons was about 62.4% and 12.8% of total population of PV-and GFP-positive neurons in laminae Ⅰ-Ⅱ of the Vc,respectively.Conclusion GABAergic neurons are mainly distributed in the laminae Ⅰ and Ⅱ of the Vc which related closely to transmission of the nociceptive primary afferent information,and the majority of PV-positive neurons are GABAergic neurons.