Establishment Of Cardiac Tissue - Specific Expression Of Neuregulin - 2 Transgenic Mice And Analysis Of Cardiac Function

Objective Neuregulins (NRGs) belong to the epidermal growth factor family mediating the signals of intercellular in the nerve system and heart. The Neuregulin-2(NRG2) is one of the members of NRGs which has been found to stimulate the developemnt of nerve system and knockout the NRG2gene can delay the embryo development. The NRG2was also expressed in the tissues of heart, but the effects of NRG2on cardiac structure and function was unknown yet. In this current paper, cardiac-specific human NRG2transgenic mice were established and the effect of NRG2on cardiac structure and function under pressure overload situation was investigated.Methods The transgenic vector was constructed by insertion of the human NRG2gene under the a-MHC promoter. The transgenic mice were generated by microinjection and were all maintained on a C57BL/6J genetic background. The genotype of transgenic mice was identified by PCR and the expression level of target gene was determined by Western Blot. Transverse aortic constriction (TAC) was applied to prepare the pressure overload induced cardiomyopathy mice model. The cardiac structure and function of the transgenic mice were compared and analysized by echocardiographic and pathological observation.Results Transgenic mice with high level of NRG2in heart tissues were established. The left ventricular wall thickness (LVPWD) was increased, and to15.6%at3months old compared with that of the non transgenic (NTG) mice. The hypertrophy of left ventricular wall caused by pressure overload was removed due to the expression of NRG2. Meanwhile, cardiac disarray and fibrosis were increased obviously compared with that of the NTG mice.Conclusion The transgenic expression of NRG2in heart tissues could shorten the pathological process of hypertrophy, but accelerated the process of heart failure (HF). Objective After the human genome project were completed, about20more thousand genes were included in the human genomic, but no more than10thousand proteins were encoded. We all know that the expression of a gene is different at the different developing stage and some gene has apparent variance in tissues and cells. It is a very important problem to solve the genes function in these tissues and cells and we need a powerful tool to analysis it. Now we have little know about the regulatory elements in the tissue and cell specificity which severely limits the gene function research process in the post-genome era. By revealing the mouse genes function can we indirectly study the human genes because of the99%homology between them. Establishing a high-throughput method screening the regulatory elements specific in tissues and cells is of great significance to study the gene function.Transposon plays an important role in the gene mutation and large scale genotype selection as a kind of genetic analysis tool. PiggyBac (PB) has higher transposition efficiency and excises tracelessly. We have established the early transposon screening system based on tissue specificity, in this study, we will use this system to screen the strain mice in tissue specific expression report gene, for the subsequent gene function study.Method Transposase transgenic mice and donor transgenic mice (F0) were mated and the double-positive F1mice were mated to wild type mice to segregate genotype in F2mice. Using the Southern Blot to dectect the changes of the insertion site before and after transposition (F2vs F0). Splinkertee PCR is used to generate the gene insert position and reporter gene expression in mammalian. X-gal staining detected the report genes position specifically.Results Southern Blot screens a high activity PB transposase founder37by mating the donor mice and transposase transgenic mice. By crossing the transposon donor mice with founder37, and separate the offspring, we screened the number of85mice whose pT-Cre-lacZ gene position was changed and the efficiency was as high as25.67%. We got4transposon mice whose pT-Cre-lacZ gene were forward insertion by Splinkertee PCR analysis. The distributon of the pT-Cre-lacZ report gene was concentrated in brain using the X-gal staining. We successfully obtained a clear gene expression profiling and genomic loci precisely mouse.Conclusion We established a high activity transposase mouse. Using the transposon system, we achieved the aim of screening the gene regulatory element specifically in tissues and cells. We successfully obtained a clear gene expression profiling and genomic loci precisely mouse. Next, we need to analysis the tissue specificity of Cre, compare the expression consistency between Cre activity and report genes.