ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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 .
ABSTRACT
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.