1.Construction of the Pkd1 gene knockout mouse model with polycystic kidney disease based on CRISPR/Cas9 technology
Guangqiang MA ; Rongliang WANG ; Ling NIU ; Hongjiao WAN ; Chenghua YAN ; Liyuan WANG
Chinese Journal of Nephrology 2023;39(9):697-704
Objective:To establish a conditional knockout mouse model of polycystic kidney disease 1 ( Pkd1) gene based on CRISPR/Cas9 and Cre-loxP gene editing technology, and to provide an animal model for in-depth research on the role of Pkd1 gene in the development of polycystic kidney disease. Methods:In-Fusion technology was used to construct a targeting vector. Corresponding gRNAs, Cas9 mRNAs, and donor vectors carrying the loxP site were prepared based on the Pkd1 gene, and injected into the fertilized eggs of C57BL/6N mice. The fertilized eggs were transferred to the fallopian tubes of female mice with pseudopregnancy. After the newborn mice were identified by PCR and sequencing analysis, Pkd1 flox/flox F0 generation positive mice were selected. The F0 generation positive mice were bred with wild-type mice, and F1 generation heterozygous mice with Pkd1 flox/+ genotype were selected for offspring. F2 generation homozygous mice with Pkd1 flox/flox genotype were obtained through internal expansion, and then hybridized with Cre positive Ggt1/ Cre mice. F3 generation mice with Pkd1 flox/+Ggt1 Cre genotype were obtained. F4 generation mice with Pkd1 flox/flox Ggt1 Cre genotype were obtained by self crossing or backcrossing with F2 generation Pkd1 flox/flox, namely kidney-specific Pkd1 gene knockout mice ( Ggt1-cKO mice). PCR method was used to identify the genotype of mice, and then the mice were divided into wild-type control (WT) group ( n=6), Pkd1 homozygous control (PKD) group ( n=6), and Ggt1-cKO knockout validation (CKO) group ( n=6) according to the gene identification results. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of Pkd1 mRNA in the kidneys and other organs of mice in each group. HE staining was used to detect the pathological changes in renal tissues of mice in each group. The automatic biochemical detector was used to detect the blood urea nitrogen and serum creatinine levels of mice, and the kidney coefficient was calculated. Results:The PCR detection results showed that the genotype of offspring mice in CKO group was consistent with Pkd1floxflox Ggt1 Cre. Pkd1 gene was only specifically expressed in the kidney, but not in other tissues. The RT-qPCR results showed that the relative expression of Pkd1 mRNA in the renal medulla of CKO group was significantly lower than that of WT and PKD groups. The kidney volume of the CKO group had increased by about twice compared to the WT group. Under the microscope, it could be observed that there were multiple vacuoles of varying sizes and shapes in the kidneys of the CKO group, and there was a significant increase in the interstitial space of the medullary tissue. The kidney coefficient, blood urea nitrogen, and serum creatinine in the CKO group were significantly higher than those in the WT and PKD groups (all P<0.05). Conclusion:Based on CRISPR/Cas9 and Cre-loxP gene editing technology, Pkd1 gene kidney conditional knockout mice can be successfully constructed, providing an animal model for further studying the action mechanism of Pkd1 gene in polycystic kidney disease.
2.Effect of Wendantang on Expression of TrKB and CREB in Hippocampus of Rats with Schizophrenia
Yuan ZHANG ; Jinhua ZHU ; Milan ZHANG ; Jing CHEN ; Hongjiao WAN
Chinese Journal of Experimental Traditional Medical Formulae 2023;29(3):26-33
ObjectiveTo study the effects of Wendantang on the expression of receptor tyrosine kinase receptors B (TrkB) and cyclic adenosine monophosphate response element binding protein (CREB) in the hippocampus of rats with schizophrenia (SCZ). MethodFifty-four rats were randomly divided into a normal group, a model group, high (40 g·kg-1), medium (20 g·kg-1), low-dose (10 g·kg-1) Wendantang groups, and a clozapine (0.02 g·kg-1) group, with 9 rats in each group. All groups of rats were given corresponding drugs by gavage for 21 d. The normal and model groups were given equal volume of normal saline. Except the normal group, the other 5 groups were given 0.6 mg·kg-1 dizocilpine maleate (MK-801) at 2 hours after the last administration for intraperitoneal injection to induce the rat model of SCZ. The level of spontaneous activity of rats was observed by open field experiment. Histomorphological changes in the hippocampal CA1 area was observed by hematoxylin-eosin (HE) staining. The protein expression levels of TrKB, phosphorylated-TrKB (p-TrKB), CREB, and phosphorylated-CREB (p-CREB) were detected by Western blot. The protein expression levels of TrKB and CREB in the hippocampal CA1 area were observed by immunohistochemistry. The mRNA expression levels of TrKB and CREB in the hippocampus were detected by real-time quantitative polymerase chain reaction (Real-time PCR). ResultAs compared with the normal group, the level of spontaneous activity in the model group was significantly increased (P<0.01). The arrangement of neuronal cells in the hippocampus CA1 area was loose and disorganized, and some neurons were denatured in the model group. The protein expression levels of TrKB, p-TrKB, CREB, and p-CREB in the hippocampus, the protein expression levels of TrKB and CREB in the hippocampal CA1 area, and the mRNA expression of TrKB and CREB in the hippocampus were significantly decreased (P<0.05, P<0.01). As compared with the model group, the level of spontaneous activity in all Wendantang groups were significantly decreased (P<0.01). The neuronal cells in the hippocampal CA1 area of rats in all Wendantang groups were relatively neatly and closely arranged, the morphology of cell body was more regular, and neurons degeneration was rarely observed. The protein expression level of TrKB in the hippocampus was significantly higher in the medium and low-dose Wendantang groups (P<0.05). The protein expression levels of p-TrKB, CREB, and p-CREB in the hippocampus were significantly higher in all Wendantang groups (P<0.05). The protein expression of TrKB in the hippocampal CA1 area was significantly increased in all Wendantang groups (P<0.05, P<0.01). The protein expression of CREB in the hippocampal CA1 area was significantly increased in the medium and low-dose Wendantang groups (P<0.01). The mRNA expression of TrKB in the hippocampus was significantly increased in the high and low-dose Wendantang groups (P<0.05, P<0.01). The mRNA expression of CREB was significantly increased in the medium and low-dose Wendantang groups (P<0.05, P<0.01). ConclusionWendantang can prevent the occurrence and development of SCZ and cognitive impairment by decreasing the level of spontaneous activity, improving the pathological damage of hippocampal neurons, and increasing the expression levels of TrKB, p-TrKB, CREB, and p-CREB.