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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.
RESUMEN
Type 2 diabetes mellitus （T2DM） is a heterogeneous disease with insulin deficiency and insulin resistance （IR） as the main etiology and is often accompanied by complications. Volatile oil is a volatile oily liquid extracted from natural plants， which has many pharmacological effects such as regulating Qi， relieving pain， inhibiting bacteria， and reducing inflammation. In recent years， there have been numerous reports on the treatment of T2DM by natural plant volatile oil and its effective components， which has become one of the new directions in the treatment of T2DM. With natural plant essential oil and its active components as the starting point， this paper comprehensively analyzed and summarized the material basis， mechanism， and signaling pathways of essential oil in the treatment of T2DM and its complications in China and abroad in recent years， and focused on the inhibitory effect of essential oil and its active components， such as carvacrol， paeonol， and β-caryophylene， on IR to improve T2DM by protecting pancreatic β-cells， inhibiting α-glucosidase activity， regulating the abundance and diversity of intestinal microbiota， and regulating glucose transporter protein type4 （GLUT4）， adenylate 5′-monophosphate-activated protein kinase （AMPK）， phosphatidylinositol-3 kinase （PI3K）/protein kinase B （Akt） signaling pathways to provide some references for the volatile oil intervention in T2DM and the development of new green antidiabetic drugs.