Studying The Biological Functions Of PZR By Using Knockout Mice

PZR(protein zero related) is a glycoprotein discovered, isolated and purified, cloned and named by Professor Zhao Zhizhuang in 1998. It belongs to immunoglobulin receptor family, and is a specific binding protein and substrate of protein tyrosine phosphatase SHP-2. Human PZR is encoded by the MPZL1(myelin protein zero-like 1) gene, and is widely expressed in tissues such as heart, placenta, kidney, pancreas and muscle. Its extracellular region contains an immunoglobulin-like domain, sharing 46% homology with myelin protein zero, and thus is named as protein zero related. The intracellular part of PZR contains two immunoreceptor tyrosine-based inhibitory motifs(ITIMs), with Tyr 241 and Tyr 263 as the main sites of tyrosine phosphorylation. When Tyr 241 and Tyr 263 residues in the intracellular ITIMs of PZR are phosphorylated, they can specifically bind with two SH2 domains of SHP-2, recruiting and activating SHP-2.Currently, domestic and foreign studies on the structure and function of PZR are very limited. Overall, the biological functions of PZR are still unclear. As we know, PZR is a specific interacting protein of SHP-2, and abnormality of SHP-2 is closely associated with various malignant diseases. It has been reported that SHP-2 was detected in about 50% of Noonan syndrome patients and approximately 90% of leopard syndrome patients. It is worth mentioning that Noonan syndrome and leopard syndrome have very similar phenotypes(mental retardation, short stature, facial deformity, congenital heart disease and skeletal muscle abnormality), but they are caused by activation and inactivation mutations of SHP-2, respectively, which suggests that two syndromes may be associated with the same signaling pathway. In addition, the activation mutation of SHP-2 occurs in 35% of juveniles with monocytic leukemia, 4% of patients with acute myelogenous leukemia and a number of patients with other cancers such as lung, stomach, colon, brain and breast cancers. PZR is discovered as a specific binding protein and substrate of SHP-2, and thus PZR as an upstream specific binding protein of SHP-2 is very likely to play important roles in the generation and development of above diseases.Gene targeting technology has been developed since 1980 s, which greatly promoted biological studies and pharmaceutical development. Gene research gradually developed from basic mapping and sequencing to research of gene function and analysis of pathogenic mechanism, which led to rapid advances in medical field and biological field. As a member of gene targeting technologies, TALEN has advantages including high targeting efficiency, low off-target effect, simple preparation and low cost, and thus it was chosen as an ideal gene targeting tool for this study.With C57BL6 as the genetic background, the present study used the TALEN technology to establish PZR knockout mouse model for the first time, developed molecular biological methods such as PCR and Western Blot to confirm successful establishment of model, and employed fluorescent quantitative RT-PCR to determine the expression level of specific gene in different tissues. Changes in the body weight, appearance and histomorphology of mice after PZR knockout were observed. Gene expression changes in the ovarian tissues of PZR +/+ and PZR-/- mice were analyzed using mouse transcriptome microarray. The present study provided basis and a very valuable animal model for systematic research on the biological functions of PZR as well as the prevention and treatment of associated diseases.1. Establishment and phenotype analysis of PZR knockout mouse modelThe open reading frame sequence of MPZL1(m MPZL1) in C57BL6 mouse is 2356 bp, containing 6 exons and encoding 270 amino acids; the open reading frame sequence of human MPZL1(h MPZL1) is 5026 bp, containing 6 exons and encoding 269 amino acids. Focused on PZR protein coding gene MPZL1, the experiment selected MPZL1-exon2 as knockout block, and deletion of target base would lead to premature termination of PZR translation.(1) At the gene level, PCR, SSCP and DNA sequencing were used to distinguish PZR +/+, PZR +/- and PZR-/- mice. At the protein level, Western Blot was used to determine expression changes of PZR protein in each tissue of PZR-/-mice. Fluorescent quantitative PCR was used to determine the m RNA expression of MPZL1 in the ovarian tissues of PZR-/- mice.(2) Mating of PZR heterozygous mice produced a large number of PZR +/+ and PZR-/- mice. Body weight, appearance and blood parameters of these mice were measured. It was found that there was no significant difference between PZR-/- mice and PZR +/+ mice in their appearance. Since weaning in the third week, the body weight of PZR-/- mice was significantly less than that of PZR +/+ mice during the same period, but there was no significant difference in their femur and organ tissue parameters. In addition, determination of blood lipid parameters showed that the fasting cholesterol and triglyceride contents of PZR-/- mice were significantly lower than those of PZR +/+ mice(p<0.05). Paraffin and HE staining were used to observe the morphology and structure of each tissue in PZR wild type and knockout type mice, developmental retardation of testicular and ovarian tissues in reproductive system was observed in PZR knockout mice, and varying degrees of hepatocyte edema was observed in liver tissues.2. Study on gene expressions in PZR-/- mice(1) Microarray technology was used to analyze the difference between female PZR-/- mice and PZR +/+ mice in transcriptome expression of ovarian tissues. It was found that the expressions of some genes associated with cholesterol and triglyceride metabolic pathways were significantly up-regulated. These include Insig1, Srebf1 and Fasn.(2) Using Western Blot, we further verified that the expressions of INSIG1 and FASN proteins in liver tissues of PZR-/- mice were all higher than those of PZR +/+ mice. How these proteins interact with PZR still requires further study.Our preliminary study on the biological functions of PZR by knockout mice provided a basis for further investigating and revealing the biological functions of PZR.