Establishment and Identification of Cardiomyocyte-specific High Expression Human APE1 Transgenic Mouse

The aim of this study is to establish transgenic mice that specifically over-express human APE1 incardiac, and to provide a tool animal for studying the relationship between the function and mutation of hAPE1 gene and cardiac development and cardiovascular diseases. A cardiomyocyte-specific hAPE1 transgenic construct containing the α-myosin heavy chain (α-MHC) promoter and human A PE1 (hA PE1) gene was generated. The transgenic vector was constructed by insertion of hAPE1 gene under the α-MHC promoter. The transgenic mice were generated by fertilized egg microinjection followed by embryo transplantation and were all maintained on C57BL/6J genetic background. The genotype of transgenic mice was identified using PCR and the expression levels of hAPE1 in different tissues were detected by Western blotting. The results indicated that cardiomyocyte-specific hAPE1 transgenic construct containing the α-myosin heavy chain (α-MHC) promoter and human APE1 (hAPE1) gene were introduced into fertilized zygotes by microinjection, and then the fertilized zygotes were implanted into the oviduct of female mice, establishing a heart specific high expression hAPE1 transgenic mouse line. 40 offspring were obtained and 15 mice carrying the human APE1 gene was identified by PCR. The heart-specific overexpression of hAPE1 was confirmed by Western blotting assay. The present study successfully obtained cardiomyocyte-specific hAPE1 transgenic mice transgenic mice expressing, which provided a useful tool for studying the function of genes in heart development and cardiovascular disease. and the transgenic mice with cardiac specific overexpression of hAPE1 were obtained. Real-time quantitative PCR and Western blotting were used to detect the expression of mRNA and protein in hAPE1 in the heart of transgenic mice. It was found that the expression level of mRNA and protein in hAPE1 in the heart of transgenic mice increased significantly. Our research showed that we have successfully constructed α - MHC-hAPE1 transgenic mice, which would provide an ideal experimental animal model for studying the detailed mechanism of hAPE1 gene in heart development and cardiovascular disease.

Keywords APE1, Transgenic, Heart, Mouse, Tissue specific Human AP-endonuclease1 (hAPE1) is not only closely related to the occurrence and development of tumors (Choi et al., 2016), but also closely related to cardiovascular diseases such as hypertension, coronary heart disease, myocarditis and cardiomyopathy (Guo et al., 2016a;Jin et al., 2017). However, its specific molecular mechanism in heart development and cardiovascular diseases has not been reported yet. Therefore, it is necessary to construct cardiomyocyte-specific high expression human hAPE1 Transgenic Mouse.
In this study, hAPE1 was constructed on a cardiac specific promoter αmyosin heavy chain (α -MHC) vector, which was injected into the nucleus of the fertilized eggs of C57BL / 6 female mice by pronucleus microinjection, and then transferred into the oviduct of the surrogate rats on the day of embolus, and the transgenic mice with cardiac specific overexpression of hAPE1 were obtained. Real-time quantitative PCR and Western blotting were used to detect the expression of mRNA and protein in hAPE1 in the heart of transgenic mice. It was found that the expression level of mRNA and protein in hAPE1 in the heart of transgenic mice increased significantly. Our research showed that we have successfully constructed α -MHC-hAPE1 transgenic mice, which would provide an ideal experimental animal model for studying the detailed mechanism of hAPE1 gene in heart development and cardiovascular disease.

Construction of cardiac specific expressed hAPE1 transgenic α -MHC-hAPE1 vector
The transgenic vector of myocyte specific expression hAPE1 constructed by 5.5 kb α -MHC gene promoter and 1 563 bp hAPE1 gene element was identified by restriction endonuclease digestion, and the result was in accordance with the expectation (Figure 1).

Establishment of the first mouse of α -MHC-hAPE1 transgene
The target fragments including α -MHC gene promoter and hAPE1 gene were purified and diluted to 2.0 ng / L by TE, then introduced into the male pronucleus of mouse fertilized eggs by pronucleus microinjection, and the viable and well-formed fertilized eggs were transplanted into the oviduct of the pseudopregnant female mouse. In this study, 40 offspring mice were born, 15 positive mice carrying hAPE1 gene were screened by PCR. The hAPE1 transgenic mice were identified by PCR ( Figure 2).

Specific expression of hAPE1 in the heart of transgenic mice
In order to verify the tissue specificity of hAPE1 gene in transgenic mice, we extracted the proteins from the heart, liver, spleen and lung of the transgenic mice, and measured them by Western blotting with the polyclona l  3,8,9,13,15,16,17,19,20,25,26,28,29,30: Mouse number antibody of APE1. The results showed that the specific bands of hAPE1 gene only appeared in the heart of the transgenic mice ( Figure 3).

Discussion
APE1 (Apurinic / apyrimidinic endonuclease 1) is a multifunctional protein, also known as redox effector factor-1. It not only has the function of repairing DNA oxidative damage, but also is the key enzyme of DNA base excision repair pathway, and can regulate the redox state in cells. It can directly or indirectly regulate redox related transcription factors (such as Nrf2, TFAM, etc.) through its cysteine residue (Guo et al., 2016b;lirussi et al., 2016;Zhang et al., 2016). In addition, in recent years, APE1 has been found to have anti-inflammatory effect in specific environment. APE1 in mammalian cells exerts more than 95% of AP endonuclease activity (Wang et al., 2014;Nassaur et al., 2016). APE1 is closely related to the incidence and prognosis of liver cancer, ovarian cancer, breast cancer, lung cancer, bladder cancer, pancreatic cancer and osteosarcoma, as well as the incidence and severity of stroke, neurodegenerative diseases and coronary heart disease (Jin et al., 2015;Stetler et al., 2016).
Human AP-endonuclease-1 (hAPE1) is not only closely related to the pathogenesis of tumor, but also closely related to cardiovascular diseases such as hypertension, coronary heart disease, atherosclerosis, myocarditis and cardiomyopathy (Jin et al., 2015;Choi et al., 2016;Guo et al., 2016a;Stetler et al., 2016;Jin et al., 2017). All of these suggest that APE1 may participate in the development of heart and these pathophysiological processes. However, the specific mechanism of APE1 in the development of heart and related cardiovascular diseases has not been reported yet. Therefore, this project has constructed transgenic mice of specifically expressing hAPE1 in heart tissue to study its role in the development of heart and related heart diseases.
In this study, heart specific expressed hAPE1 transgenic mouse was constructed by using the transgenic technology. The offspring mice integrated with the hAPE1 gene were screened by PCR and agarose electrophoresis. The detection results showed that in the mice confirmed as positive by PCR, the expression of hAPE1 in the transgenic mice increased only in the heart, suggesting that the heart specific hAPE1 transgenic mouse was successfully constructed. In order to avoid the possible influence of overexpression of hAPE1 on the embryonic development of the heart, in this experiment, we selected a specifically expressed α-MHC promoter in cardiomyocytes, which was convenient to study the role of hAPE1 in non-congenital heart diseases (such as myocarditis, cardiomyopathy and ischemic heart disease). The heart-tissue specific hAPE1 transgenic mice established in this study would provide a good animal model for the study of the pathogenesis of heart disease and drug screening about human.

Plasmid, toolenzyme and reagent
The plasmids containing alpha-myosin heavy chain (α-MHC) promoter of cardiomyocytes specifical expressed Figure 3 Western blotting measured the expression of hAPE1 in liver, spleen, heart, lung, and kidney Note: TG76 and TG80: Positive mice proved by PCR; NTG182 and NTG: Negative mice proved by PCR; WT: Wild type mice were donated by Dr. Jeffrey Robbins (University of Cincinnati, Cincinnati, OH, USA). The wild-type expression plasmid of human APE1 (hAPE1) was donated by Dr. Zhiqing Wang of Johns Hopkins University (Department of Pediatrics, Baltimore, USA). All plasmids were analyzed by restriction endonuclease digestion and electrophoresis, and sequenced at the end of the plasmids. The plasmids were confirmed by searching and comparing on the Internet. DH5α strain was preserved in our laboratory.
Restriction endonucleases, T4 DNA ligases, Tag DNA polymerases, SP6 RNA polymerases, T3 RNA polymerases used were bought from New England Biolabs, Promega and TaKaRa, protease K from Merck, dNTP from Pharmacia, RNase A, RNase T1 from Boehringer Mannheim, mRNA Selective PCR Kit from Takara, X-gal, IPTG, Primer-A-Gene Labeling System was purchased from Promega.
RPMI1640 medium and DMEM medium were purchased from Hy-CIone company, plasmid mass extraction kit was product of Tiangen company, APE1(#4128) and β-actin antibody were purchased from Cell Signaling Technology company.

Experimental animals and feed
Clean grade C57BL/6J mice and special feed for mice were purchased from Guangdong Medical Experimental Animal Center and raised in SPF experimental animal room of Guangdong Medical University. They could drink clean water and eat freely except as indicated. Healthy and clean C57BL/6J mice, male and female were unlimited. All animal experiments were conducted in accordance with the regulations of Guangdong Medical University on the administration of experimental animals.

Construction of α-MHC-hAPE1 transgenic vector
The Gateway technology was used to construct the vector. The eukaryotic expression vector pPB (Exp) -alphaMHC long > hAPEX1 (Vector ID: VB160929-1104yhc) was used as the template for PCR amplification. The target fragment of hAPEX1 gene (NM_001641.3) was obtained. BP reaction, LR reaction, plasmid purification and linearization were carried out.

Establishment of α-MHC-hAPE1 transgenic mice
The plasmid was purified and linearized, and then injected into the uterus of the pseudopregnant female mouse by microinjection of the fertilized egg and embryo transfer. The fragment was introduced into the male pronucleus of mouse fertilized eggs. 40 offspring mice were obtained. The tail DNA of offspring mice was extracted for PCR detection and analysis. The 0.5 cm tail tip of mice was cut after 7 days of birth and placed in a 1.5 mL centrifuge tube. 400 μL tissue lysate was added and spent at 55 ℃ overnight. PCR amplification and electrophoretic detection were carried out with α-MHC-hAPE1 transgene specific primer to screen the offspring of integration of external genes. The hAPE1 positive mice could have 689 bp internal reference bands and targeted bands with the size of 385 bp / 466 bp / 391 bp. PCR results showed that there were two bands in 15 mice, carrying the foreign gene of hAPE1 ( Figure 2). Primers (synthesized by Shanghai Biotechnology Co., Ltd.) include primer 1: F1: GAAGTGGTGGTGTAGGA AAGTCAG, R1: GCTGGTAGTTTGTTCTCTGAAC, primer 2: F2: CTTCGGGGAATTACTGCAGGCT, R2: GAACTTGTGGCCGTTTACGTCG, primer 3: F3: AACTTCAAGATCCGCCACAACA, R3: AGAAGTCAGA TGCTCAAGGGGC, internal reference primer F: GCAGAAGAGGACAGATACATTCAT, R: CCTACTGA AGAATCTATCCCACAG, and expected PCR. product size: 466 bp, 385 bp, 391 bp. The target fragment of PCR is the mouse endogenous Rgs7 (G protein signaling 7). The expected PCR product size is 689 bp. 38 cycles were under the standard condition of 25 μL PCR reaction system. Taq DNA polymerase was used with Takara R004A. There was no DNA template in the water control group, 400 ng genome DNA of wild-type mice in the wild-type control group, and the 400 ng genome DNA of mice containing injected DNA in the positive control group (equivalent to the genome of diploid mice, containing 5 copied transgenic fragments). All the PiggyBac transgenic mice with positive target genes did not contain the integration of auxiliary plasmids by being verified by PCR. PiggyBac assisted plasmid primer-F: CTGGACGAGCAGAACGTGATCG, R：CGAAGAA GGCGTAGATCTCGTCCTC, and the size of expected PCR product was 352 bp.

Detection of APE1 expression in heart tissue of α -MHC-hAPE1 transgenic mice
In order to verify whether the target gene hAPE1 is Characteristic expression in the heart tissue of transgenic mice, we randomly selected a female and a male from the constructed mice, and produced F2 generation through mating, forming two independent APE1 transgenic mouse lines. The cardiac tissue proteins were extracted from the heart, liver, spleen, lung and kidney of wild-type and transgenic mice respectively. Through Western blotting analysis, it was found that APE1 protein level in the transgenic mice of two lines (Line1, Line2) was up-regulated compared with that of wild-type mice, and the expression level in the cardiac tissue was significantly increased compared with other tissues (Figure 3), indicating that the transgenic mice of heart tissue specific overexpressing APE1 were successfully established.