Clone Of Bmal1Gene Promoter In Mice And Its Construction Of Luciferase Reporter Gene Vector

Objectives: Bmal1gene aka MOP3or ARNTL (aryl hydrocarbonreceptor nuclear translocator like protein), an important transcription factor,was successfully cloned in a lab in1997. Bma1gene contains structuraldomain of Per-ARNT-Sim(PAS)�'�bHLH(basic Helix-Loop-Helix), and couldfunction as a transcription factor to regulate gene transcription in combinationwith DNA. Bmal1homologous genes exist in a variety of organisms amongwhich humankind’s Bmal1gene is located on Chromosome11, while mice’ onChromosome7, rats Bmal1gene on Chromosome1. It is found out that Bmal1is most active in transcriptionduring the segment from upstream816bp to thedownstream99bp. Sequence alignment found out that this section contains anumber of transcription factor binding sites, such as SP1, AP1, NF-Y and soon. Subsequent studies have found that Rev-erb alpha, Rev-erb beta, theRORa, RORb is vital for Bmal1transcription factor is combined with such asin the area. The latest research shows that DNA methylation in Bmal1plays akey role in regulatory function in Bmal1transcription. Regulated bymethylation, CpG island is also located on the above-mentioned915bp section.Therefore, the915bp section is widely used in study of Bmal1genetranscription activity.Bmal1plays an important role in many aspects. Firstly, Bmal1ismolecular basis of circadian rhythm. Secondly, Bmal1could decide agingprocess by regulating oxidative stress. Thirdly, Bmal1has important immuneregulating function. Fourth, Bmal1takes part in regulation of sugarmetabolism. Fifth, Bmal1also take s part in the activity of regulating femalereproductive ability. In addition, Bmal1is associated with tumor development.Knocking out and knocking down Bmal1gene can both exert an effect onphysiological function at multiple levels. What is interesting is that Bmal1 gene does not express on all tissues or cells. This research in the laboratoryshows that embryonic stem cells showed heterogeneity in Bmal1expressionlevel: a part of the cells are Bmal1gene positive, while another part negative.This heterogeneity can be seen in the thymus and testicles as well. Currentlythere is no effective way to separate and make a selection among Bmal1positive and negative cells for further study. This research aims at solving thistechnology bottleneck by using genetic engineering approach and constructionof pLV-Bmal1-mCherry-EF1-copGFP-T2A-puro double fluorescent carrier. Inthis way, we could study the biological features of Bmal1promoter in livingcells and tissues in a convenient way.Methods: We firstly identify the section of Bmal1gene promoter,which starts transcribing from upstream of transcription position-816base to downstream of the transcription-99base. The whole length covers915bases. This section contains combining position of a number of transcription factors, including SP1, AP1, NF-Y。Rev-erb alpha, Rev-erb beta, RORa, RORb and other factors that all play an irreplaceable role inthe transcription of Bmal1gene in this section. It is also in this sectionthat methylation happens as a regulatory role in Bmal1transcription. This915bp section is widely used in the study of Bmal1gene transcription. We use high fidelity enzyme and mice’s liver genomic DNA as a template to amplify Bmal1promoter sequences. By double enzyme, we cloned Bmal1gene promoter into lentiviral vector pLV-CMV-MCS-EF1-copGFP-T2A-puro, which resulted in the middle clone of pLV-Bmal1-MCS-EF1-copGFP-T2A-puro. And again by double enzyme, we clone redfluorescent protein（mCherry）into pLV-Bmal1-MCS-EF1-copGFP-T2A-puro, which resulted in the final goal of cloning pLV-Bmal1-mCherry-EF1-copGFP-T2A-puro. We used this clone and pLP1、pLP2、pLP3to transfect a HEK293cell together and then packaged them into a virus. We obtained the virus and concentrated it with super centrifugal purification.We examined the virus titer of the obtained infected HEK293cell.Results: We obtained the Bmal1gene promoter segment with the mice’ genomic DNA as template. The ultimate clone of pLV-Bmal1-mCherry-EF1-copGFP-T2A-puro after two successful steps of cloning provedto be right in structure if examined by Enzyme digestion and sequencing. There are two systems of reporter genes: the red fluorescent proteintranscription is controlled by Bmal1promoter; green fluorescent proteintranscription is controlled by constitutive promoter EF1. We draft thisdesign in consideration that the efficiency rate of infecting a cell withlentivirus may not reach100percent. Due to that EF1could be expressed in almost all kinds of tissues and cells, cells will express green fluorescent protein and give a green glow if the virus successfully infectsthe target tissues and cells, while uninfected cells will not express fluorescent protein and have no green glow. Only the cells with green glowis recognized as a successfully virus infected cell and will be analyzedfurther on the expression of red fluorescent protein-which is the expression of Bmal1gene. This design prevented the false negativity sign in the process of separation and making a selection. Virus-infected HEK293cells packaged with this clone shows a titer of3*108TU/mL in examination, and can be used in follow up infection experiment.Conclusion: This experiment successfully structured a lentivirus vector with red fluorescent protein which is regulated by Bmal1promoter, and obtained virus with high titer. It laid a solid foundation for differentiating Bmal1positive cells and Bmal1negative cells and analyzing similarities and differences between them in terms of their biological characteristics.