eNOS基因敲除大鼠,动物造模 z-bio 2021-05-17 383

　　Hypertension is a complex polygenic disease characterized by increased arterial blood pressure. It is one of the main risk factors for coronary heart disease, myocardial infarction, and stroke. Its morbidity and mortality are increasing year by year, which is a serious threat to human beings. health. The occurrence of hypertension is related to increased secretion or increased activity of vasoconstrictor substances, and decreased secretion or decreased activity of vasodilatory substances. Endothelial nitric oxide synthase (eNO S) plays an important role in regulating blood pressure. Vascular endothelial cells produce nitric oxide (N0), which acts on vascular smooth muscle cells to cause vasodilation, which ultimately leads to a decrease in blood pressure. The establishment of an eNOS gene knockout rat model can lay a foundation for the study of the pathogenesis and treatment of hypertension.

　　1 Materials and methods

　　1.1 Animals

　　SPF grade SD rats, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. [SC X K (京)20 12.0001]. All experimental mice were raised in the Laboratory Animal Department of China Medical University [SY X K (~ )2013.000 1], the temperature was 24 ± 2 ℃, and the relative humidity was 50% ± 10%. Automatic light control (12h bright, 12h dark) [SY X K (Liao) 20 13-0007].

　　1.2 Preparation of eN O S gene knockout rats

　　1.2.1 Purification of the injected gene Using zinc finger nuclease (Z FN) technology, the knock-out vector was constructed, and the restriction digestion was linearized, and the gate SSA G E m M A C H IN E of Ambiion was used. T7 U L TR A K it was transcribed in vitro and then purified by Qiagen's R nesay Mini K it.

　　The purified fragments are dissolved in water and stored at -80°C. Dilute before injection, and the injection concentration is 10 ng/~tL.

　　1.2.2 Preparation of superovulation and recipients in rats Choose 5-week-old SD rats by intraperitoneal injection of PM SG 25 IU/mouse, 46" -~48 hours later, intraperitoneal injection of hCG 25 IU/mouse, the same day as normal SD male rats 1: 1 mate in the cage, check the vaginal plug the next morning, and the pregnant mouse with the plug as the donor mouse. While superovulating the cage, select wild-type SD female rats to mate with the ligated SD male rats. In the morning, the vaginal plug was checked, and the pregnant mouse with the plug was seen as the recipient mouse.

　　1.2.3 Collection of fertilized eggs The female donor mice were sacrificed by cervical dislocation method, the fallopian tubes were collected, placed in M 2 culture drops preheated at 37 ℃, and the enlarged part was torn under a stereo microscope to obtain one oocyte and one egg The cumulus cell complex was treated with 1 mg/mL hyaluronidase to obtain a single fertilized egg, washed in M 2 for 3 to 5 times, and then recorded the number of fertilized eggs, and transferred to K SO M medium incubated overnight. To be injected.

　　1.2.4 Microinjection The in vitro transcription and purification of mRNA encoding eNOS-specific zinc finger enzymes were injected into rat fertilized eggs, and the fertilized eggs in good condition after injection were transferred to a CO z incubator for incubation 30 Embryo transfer is performed after min.

　　1.2.5 Embryo transfer According to 0. 1 g/m L chloral hydrate is 0. 31 ml/100 g intraperitoneal injection of SD pseudo-pregnant rats were anesthetized. After alcohol disinfection, the back kidney hair was cut, the skin was cut, and the opening was cut near the small bend of the fallopian tube, and the oviduct inserted into the fallopian tube from the cut opening. The fertilized egg is blown into the ampulla, stays for a while and then pulled out. Reset the ovary, fallopian tube, and uterus, and suture the wound.

　　1.2.6 Rat detection cut out the tail tissues of young rats from 10 to 15 days old, and digest them in a water bath shaker at 55 ℃ under the action of proteinase K. The genomic DNA of rat tails was extracted by the phenol-chloroform method, and the forward bow was designed for detection. l Object: 5'-TGTG TT CTCTC TT CTGGCTCAGG-3', reverse primer: 5'-CAAAGA TC c T rC CACGAAC. 3. Carry out the PC R reaction under the following conditions: pre-denaturation 94 ℃ 5 min, 94 ℃ 45 S, 60 ℃ 45 S, 72 ℃ 45 S, 34 cycles, and finally 72 ℃ extension 10 min. The PC R product was sequenced, T. A Clone, confirm the gene deletion situation.

　　1.2.7 Homozygous screening The primary positive mice were bred with wild-type SD rats to produce F 1 generation heterozygous rats, F 1 generation heterozygous rats mate with each other, and homozygous rats were screened.

　　1.3 W estern blot detection

　　Take the heart and kidney tissues of eNO S knock-out rats and SD wild-type rats, quickly place them in liquid nitrogen, pulverize the tissues, add protein lysate for lysis, centrifuge and take the supernatant, which is the total protein. After protein quantification and denaturation, perform SD S PA GE gel electrophoresis, transfer membrane, block in 5% non-fat milk, and incubate the antibody. The primary antibody is Immunow Ay Company antibody, and the secondary antibody is goat anti-rabbit antibody, which is subjected to ECL chemistry. After the detection reagent emits light, the imaging is performed to detect the expression of eN OS. At the same time, G A P D H is used as an internal reference.

　　1. 4 Observation of the appearance and phenotype of homozygous rats with eN O S gene knockout. The head, trunk, limbs, tail, etc. of homozygous rats and littermate wild-type rats were visually inspected to observe whether there are deformities.

　　1. 5 Observation of histology and morphology

　　The heart, liver, spleen, lung, kidney, muscle, and abdominal aorta vascular tissues were taken after the rats were sacrificed and put in 0. Fix in 25 g/ml paraformaldehyde, embed in paraffin, section, stain with hematoxylin-eosin (HE), observe the changes of tissue structure and take pictures under light microscope.

　　1. 6 Body weight comparison We selected 5 male and female wild-type SD rats, and 5 male and female eNO S knockout rats. The body weight of 8-16 weeks old was measured and compared.

　　1. 7 Determination of heart rate, blood pressure, systolic blood pressure, and diastolic blood pressure selected wild-type SD rats with 5 males and 5 males, and ⅣD knockout rats with 5 males and 5 males, 8-9 weeks old. Using the Softron BP-20 10A instrument, the rat’s heart rate, blood pressure, systolic blood pressure, and diastolic blood pressure were measured by the tail cuff method. Each rat was measured 3 times, and the average value was recorded as the rat’s heart rate, blood pressure, systolic blood pressure, and diastolic blood pressure. Pressure.

　　1. 8 Statistical analysis

　　The data are expressed as the mean ± standard deviation ± ), and the differences between groups are tested by t, P<0. 0 5 means the difference is statistically significant.

　　2 Results

　　2.1 The genotype test results of eN OS knockout homozygous rats The eN OS knockout vector was transcribed and purified in vitro, and injected into SD rat fertilized eggs. A total of 441 fertilized eggs were injected, and 365 survived, and 12 were transplanted into them. The fallopian tubes of SD rats produced a total of 23 FO rats. A total of 4 positive rats were obtained by sequencing and analysis of PCR products, among which rat No. 8 was selected for passage breeding and selection of homozygotes. The knockout-positive rat lacked 22 bases in the 4th exon, resulting in a frameshift mutation.

　　2.2 W estern blot detection

　　When GA PD H is used as an internal control, wild-type SD rats express eNOS protein in the heart and kidney, and eNOS knockout rats express eNOS protein.

　　2.3 Observation results of appearance phenotype

　　ENO S gene knockout homozygous rats showed varying degrees of limb defects.

　　2.4 Pathological observation

　　Wild-type SD rat arterial intima endothelial cells are single-layered and flat with a smooth surface, while the eNO S gene knockout rat artery intima endothelial cells are swollen, the surface is uneven, and the adventitia is damaged (Figure 4). Other organizations have no obvious changes in organizational morphology and structure. Laboratory Animals and Comparative Medicine L aboratory A ni m al and C om parat ive M edi cine A ug. 2015, 35(4)

　　Note: Compared with wild-type rats of the same sex, P<0. 05, "P<0. 0 1

　　3 Discussion

　　Rats are more meaningful in physiology, pharmacology and behavioral research, especially in certain cardiovascular diseases, behavioral research and neurological research than mice, and are ideal animal models. In terms of evolution, rats are closer to humans than mice. In some studies of inflammatory diseases and neurological diseases, rats are used as animal models, and their phenotypes are closer to human clinical manifestations. For example, in the large and mouse models of Alzheimer's disease that have been transgenic for the same gene, the phenotype of the transgenic rat is not exactly the same as that of the mouse. The transgenic rat model can effectively replicate the pathological changes of the brain in Alzheimer's disease. Its phenotype is more similar to the occurrence of human diseases, and it is more suitable as an animal model of Alzheimer's disease. Due to the characteristics of rats in physiology, behavior, metabolism, etc., the role of rats in life science research has become increasingly important. Some human diseases, such as hypertension, do not have good mouse models and must rely on the establishment of rat models. Rat genetic engineering technology plays a vital role in the development of rat models. Due to the limitation of the culture conditions of rat embryonic stem cells (ES cells), the traditional gene targeting technology based on rat ES cells has been greatly affected, which makes the gene modification technology that does not rely on ES cells show great advantages. In recent years, a variety of high-efficiency research tools have been developed, including ZFN s technology, TAL EN s technology [10 and C RISP CAS9 technology, which provide new methods for genome editing and provide new ways to study gene functions. The application of these technologies does not require the ES cell targeting stage, and the preparation cycle is short and the efficiency is high.

　　This study used ZFN technology to establish eNO S knockout rats. The rat has a deletion of 22 bases in the 4th exon, resulting in a frameshift mutation. The gene knockout homozygous rats were tested for protein expression and found that the gene was not expressed in the kidney and heart. Immunohistochemistry experiments showed the same results (results not shown), achieving the goal of gene knockout.

　　Observation of eN OS knockout rats found that homozygous knockout rats have different degrees of limb defects, while heterozygous rats have normal appearance. The size of knockout rats is smaller than wild-type rats. A comparative analysis of the body weight of older rats showed that the body weight and the increase in body weight of the knockout rats were significantly lower than that of the wild-type rats. This is consistent with the results of earlier studies on mice that knocked out eNO S. Female mice that knocked out eNO S showed a reduced number of embryos, a high rate of stillbirth, and smaller fetuses [12,13]. Knockout rats have varying degrees of limb defects, which will also have a certain degree of impact on food intake, thereby affecting the increase in body mass. eNO S is the key to regulating vascular function, and it also plays an important role in regulating the vascularization of endothelial cells [1 4, 15]. The HE staining of rat arteries in this study shows that the adventitia of the arteries is obviously damaged, which will have a certain impact on vascular function. The effect of eNO S gene on limb development was not seen in knockout mice, but the phenotype was obvious in knockout mice. The effect of this gene on limb development needs to be further studied.

　　In eNO S gene knockout mice, blood pressure was significantly increased, and both systolic and diastolic blood pressure were also significantly increased. Under normal physiological conditions of the body, vascular tension is mainly regulated by vasoconstriction factors and relaxation factors. As an important vasodilator in the body, NO, together with other vasodilators, mediates the vasodilation response, and can resist the vasoconstrictor effects of vasoconstrictors in the body, such as angiotensin II and renin. In most blood vessels, the endothelium-dependent diastolic function is mainly due to the NO product derived from eNO S. Down-regulation of eNO S can cause a large reduction in NO and weaken the vasodilator function. In the study of eN O S knockout mice, it was shown that the blood pressure of eN O S knockout mice was significantly higher than that of wild-type mice. It was proved that eN O S affects blood pressure levels in mice. A study by Savard et al. showed that eNO S gene transfer can enhance the release of NO and may prevent the exacerbation of hypertension. In addition to the increase in blood pressure, this study observed that the heart rate of the gene-knockout female mice was significantly reduced, while the heart rate of the male mice did not change significantly. This may be the response of the reflex mechanism of the baroreceptors to the increase in blood pressure. In summary, this study successfully established eNO S gene knockout mice, which can be used as an ideal animal model for studying the pathogenesis of hypertension, and at the same time lay the foundation for the study of eNO S gene function.