Construction And Identification Of Stable Cell Lines As A Long-term Uric Acid Sensor Or A Responsible System

Synthetic biology is an interdisciplinary of the post-genome era which develops rapidly, it solves several major issues facing human development by transforming organisms purposefully, bringing light for the settlement of major issues facing human such as energy, health and the environment. The construction of a genetic circuit is the most important achievement of the development of synthetic biology. Along with the rapid development of synthetic biology, more complicated and delicate genetic circuit is around the corner, bringing a breakthrough in medicine, medicine industry and agriculture areas.Purine turns into Uric acid(UA) through catabolism. The latter is slightly soluble in water, and easy to form urate crystal precipitation. In normal circumstances, uric acid homeostasis in blood has been maintained. Increased production or decreased excretion will result in higher blood uric acid levels, when the concentration of blood uric acid is higher than 420 μM, it is known as high blood uric acid(hyperuricemia). hyperuricemia can harm human health, long-term hyperuricemia causes diseases such as gout or tumourlysissyndrome(TLS)At present, gout relies mainly on drug therapy, traditional medicine and new type biological included, which both have some shortcomings.Traditional medicine has a better safety but a weaker efficiency while the new type biological on the opposite; New type biological although has a powerful therapeutic effect, easy cause allergic reactions and drug resistance. Along with the development of synthetic biology, the researchers saw huge potential of synthetic gene circuits in self-sufficient control of urate homestasis.According to literature reports, R1 in the genome of Deinococcus radioduran has been translated to transcriptional repressor huc R. Mediated by uric acid, huc R affects downstream gene expression through combination or separation to huc O, a DNA sequence motif. When functional gene sm Uox is chosen as the downstream gene, Uricase has been expressed, so that urate levels have been controlled. In the presence of uric acid, Huc R and huc O work as a“switch”. When urate levels reach a certain value, Huc R is released from huc O8 thereby inducing downstream gene expression, lowering urate levels. When urate levels are too low, Huc R binds huc O8 and silences expression of sm Uox, so that urate homeostasis has been maintained.In previous studies, Huc R has been optimized to urate-dependent transsilencer m UTs; meanwhile, we place eight tandem of huc O to get huc O8. The double-carrier uric acid sensor circuit has been constructed by cotransfected "m UTs" and "huc O8” into cells, using secretion of alkaline phosphatase(SEAP) as a report gene. To construct a single-carrier uric acid sensor circuit, "m UTs", "huc O8"and the report gene has been integrated together. As a result, whether the double-carrier uric acid sensor circuit or the single-carrier uric acid sensor circuit can reflect urate levels in the environment(0-2000 μM); besides, the proportion of m UTs/huc O8 can affect the function of the circuit.The main purpose of the study is to establish a stable cell line as a long-term uric acid sensor system at the cellular level. On the other hand, establish a stable line which can maintain urate homeostasis can at the cellular level and reduce pathologic urate levels into a physiologic window(90-420 μM). The main contents and results are following:1、Establish the stable cell line p-Huc O8-SEAP-m UTs, the relative expression of m UTs determined by q PCR.The results show that the relative expression of m UTs is 21.8 ± 2.6 times higher than control cell line while the relative expression of m UTs of transient cell line only 3.7 ± 0.15 times higher than the control. the relative expression of m UTs remaining 17.64±2.58 folds of control cell line in 2nd、4th、6th、8th、10th generations The results indicate that the stable cell line p-Huc O8-SEAP-m UTs is successfully established, which, has a great stability.2、Detect the relative expression of SEAP of the stable cell line under the different concentration of uric acid(0-800 μM).The results show that the relative expression of SEAP is raising with the increased urate levels up to 2.79±0.21 folds of the control. Change the urate level and determine the relative expression of SEAP every 24 h, the results show that the stable line can reflect the uric acid concentration of the circumstance effectively, and has a great reversibility.3、Cultivate the stable line, Change urate levels and determine the relative expression of SEAP every 24 h(0?800).The results show that The results show that the stable line can reflect the uric acid concentration of the circumstance effectively, which was not influenced by the former concentration of uric acid in circumstance. The results indicate that the stable line has a great reusability. The stable line p-Huc O8-SEAP-m UTs can serve as a long-term sensor system at cellular level.4、Construct carrier p-Huc O8-sm Uox-m UTs by using sm Uox as the downstream gene. Construct the single-carrier uric acid responsible circuit by Hep G2 transient transfection assay. Characterize its performance in controlling urate levels. The results show that transient transfected Hep G2 can regulate urate levels, that, it can reduce urate levels from 800 μM to 576.03±8.59 μM.5、Construct carrier p-huc O8-sm Uox by using sm Uox as the downstream gene. Construct the double-carrier uric acid responsible circuit based on carrier p-huc O8-sm Uox and pc DNA3.1/V5-m UT and characterize its performance. The results show that the double-carrier uric acid responsible circuit can reduce abnormal urate levels(600/800 μM) to(441 ± 0.37/635 ± 1.29 μM).But, it cannot reduce it into a physiologic window.6、Establish three stable lines p-Huc O8-sm Uox(C3, D3, and D4) based on the carrier p-huc O8-sm Uox via lentivirus transfection. p LV-m UTs-m Cherry has been transiently transfected into cell to characterize the performance of the stable line in controlling urate levels.The results show that, transiently transfected strain Trans-C3 has a better ability of maintaining urate homeostasis compared to D3 and D4,which it can reduce 800 μM to 395.4±0.37 μM within the physiologic window in 24 h...