| Objective:The Middle East Respiratory Syndrome（MERS）is another infectious disease of respiratory system discovered after SARS,with a death rate of 34.42%,caused by the Middle East Respiratory Syndrome Corona virus（MERS-CoV）.Animal models,which are susceptible to MERS-CoV and consistent with human infected performance,provide a powerful tool for the discovery and research on MERS-CoV transmission,prevention and treatment.Dromedary and non-human primate infected models,which with a high cost,are not suitable for large-scale screening.Human dipeptidyl peptidase 4（hDPP4）is the cell surface receptor of the MERS-CoV,which mediates the entering of viruses into human cell.Due to the mouse dipeptidyl peptidase 4（m DPP4）lacks the ability to bind with the Spike protein of MERS-CoV,MERS-CoV can’t infect mouse and can’t lead to any symptoms as in infected human.By introducing hDPP4 into mice,the expressed hDPP4 maybe can cause the MERS-CoV infection in mouse.So,a genetically modified mouse model may facilitate to develop a rodent model with high infected efficiency and typical clinical symptoms,which can simulate the infection in human.Now,several kinds hDPP4 transgenic mice have been tested in MERS-CoV infection,but almost all kinds of these mice lacked the growing development and serum biochemical index,which is essential for results demonstrating.We intend to construct a transgenic mouse that expressing hDPP4,named C57BL/6J-TgN（UBC-hDPP4-GFP）CLARS.It is referred to TghDPP4.The TghDPP4were identified by PCR to confirm its genotype.qPCR and Western blot were performed to detect the distribution of hDPP4.In order to provide basic data for the application of MERS-CoV in related studies,the growth and development index,organ index and blood biochemistry of mice were examined.Methods:1.Construction of C57BL/6J-TgN（UBC-hDPP4-GFP）CLARS mice.（1）Construction of pUBC-hDPP4-IRES-GFPSearch the hDPP4 gene sequence in NCBI database（Gene ID:1803,Gen Bank:NM＿001935.4）and design cloning primers according to it.Clone the hDPP4 cDNA sequence from the human cell Huh7,and insert it into pUBC-IRES-GFPtoconstructrecombinantplasmid pUBC-hDPP4-IRES2-GFP.pUBC-hDPP4-IRES-GFP was verified by restriction endonuclease digestion reaction and sequencing.（2）Construction of C57BL/6J-TgN（UBC-hDPP4-GFP）CLARSpUBC-hDPP4-IRES-GFP was linearized and injected into the pronuclear of fertilized eggs.GenomicDNA（g DNA）of offspring mice was extracted,and PCR was performed to screen out the positive offspring mice that successfully integrated hDPP4 gene,which was named C57BL/6J-TgN（UBC-hDPP4-GFP）CLARS or TghDPP4 F0.（3）Breeding of TghDPP4 mice.The confirmed TghDPP4 F0 was mated with the wild-type C57BL/6J mouse to obtain the F1 mouse.According to the results of genotyping,TghDPP4F1 mice were selected to mate with wild-type C57BL/6J female mice to breed F2 generation transgenic mice.Select TghDPP4F2 mice and use in vitro fertilization technology to breed F3 mice.Except the identification of the genotype,the TghDPP4 F3mice were also performed by qPCR and Western blot to detect the distribution of hDPP4.2.Growing development and biochemical index of TghDPP4 F3.We measured the length of body and tail of the TghDPP4 F3mice after euthanasia was performed on the TghDPP4 F3mice,the organs weight was also measured.The index was calculated.Blood was collected from the ophthalmic vein and the blood biochemical characteristics of the TghDPP4 F3mice were analyzed.Results:1.The total cellular RNA was extracted from the human cell Huh7,and was successfully reverse transcribed into cDNA to amplify the hDPP4fragment.It was connected with the vector pUBC-IRES-GFP.Blast analysis showed that the hDPP4 fragment has 100%homology with the hDPP4sequence in Genebank,and the pUBC-hDPP4-IRES-GFP plasmid was successfully constructed.2.A total of 4 F0 mice and 15 F1 mice were obtained by mating the male TghDPP4F0 mice and female wild-type C57BL/6J mice.Among them 8 mice were positive TghDPP4,with a positive rate of 53%.A total of 31 F2 mice were obtained by mating the male TghDPP4F1 mice and female wild-type C57BL/6J mice.Among them 17 mice were positive TghDPP4,with a positive rate of 55%.A total of 15 TghDPP4F3 mice were obtained through IVF technology,and the positive rate was 58%.3.The m RNA expression level of hDPP4 was higher in liver,spleen,lung and stomach tissues than in the other organs in TghDPP4F3 mice;the protein expression was higher in lung,brain,spleen,kidney in TghDPP4F3mice.4.There were no significant differences on body weight,tail index and viscera index between TghDPP4F3 mice and wild-type C57BL/6J mice.But the over expressed hDPP4 can affect the growth and development characteristics of the male TghDPP4F3 mice.The body weight of male TghDPP4F3 mice is significantly higher than that of wild-type C57BL/6J mice since 5 weeks of age.5.However,the over expressed hDPP4 affected blood glucose,blood urea ammonia,alanine aminotransferase,alkaline phosphatase in TghDPP4F3mice.The alanine aminotransferase and alkaline phosphatase of female TghDPP4F3 mice is higher than wild-type C57BL/6J mice.The alanine aminotransferase and alkaline phosphatase of male transgenic mice is lower than the wild-type C57BL/6J mice.Elevated blood glucose and blood urea ammonia were found in male TghDPP4F3 mice.Conclusions:1.The transgenic mice expressing hDPP4 were successfully constructed,and the exogenous gene can be inherited and stably expressed in the transgenic mice.2.Through the analysis of the growth and development characteristics,viscera index,blood biochemistry of TghDPP4F3 mice,it is known that the exogenous hDPP4 gene have no effect on the body length,tail length and viscera index in that mice.But it can affect the growth and development characteristics of male TghDPP4F3 mice.And the exogenous hDPP4 can affect the blood sugar,blood urea ammonia,alanine aminotransferase and alkaline phosphatase in transgenic mice. |
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