Screening For Enhancer Elements Of Conserved Non-coding Sequences Of SHOX Gene

Objective：To analyze the specific location, quantity of conservativenon-coding elements (CNEs) of short stature homeobox containing gene(SHOX). To identify these CNEs and screening for enhancer of SHOXgene with enhanced activity.Methods：1.Comparative genomics analysis and screen all CNEs of PRA1of SHOX gene.2.To construct green fluorescent protein plasmids ofcontaining different CNEs.3.These plasmids transfect HEK293cells(human embryonic kidney cells),and extract total RNA.4.To analyzerelative quantity of mRNA of SHOX gene of various CNEs transfectioncells with Real-time Quantitative PCR Detecting System.5.And analyzedatas with statistical method.Results：1. Non transfection group (not to make any intervention cells) and controlgroup (add empty plasmid without CNEs), P=0.96(>0.05), no statisticaldifference.SHOX gene expression of non transfection group and controlgroup have no distinguish. 2. CNE-4, CNE2, CNE3, CNE4, CNE5, CNE6, CNE7, CNE8, CNE12,CNE13, CNE14, CNE15compare with non transfection group,and obtainthat the P are0.706,0.942,1,1,0.416,0.109,0.416,0.838,1,0.108,0.866,0.932(>0.05).SHOX gene expression of CNE–4,CNE2,CNE3,CNE4,CNE5,CNE6,CNE7,CNE8,CNE12,CNE13,CNE14,CNE15are identicalto non transfection group.3.CNE-5,CNE-3,CNE-2,CNE9,CNE10,CNE11compare with nontransfection group,and obtain that the P are <.0001,<.0001,<.0001,0.015,0.01,0.007(<0.05).SHOX gene expression of CNE-5,CNE-3,CNE-2,CNE9,CNE10,CNE11are different from non transfection group.AndSHOX gene expression of these groups is higher than non transfectiongroup.Conclusion：To use bootstrap on multiple comparisons in proportions:1. Non transfection group (not to make any intervention cells) andcontrol group (add empty plasmid without CNEs), P=0.96(>0.05), nostatistical difference.SHOX gene expression of non transfection group andcontrol group have no distinguish.2. CNE-4, CNE2, CNE3, CNE4, CNE5, CNE6, CNE7, CNE8,CNE12, CNE13, CNE14, CNE15compare with non transfection group,andobtain that the P are0.706,0.942,1,1,0.416,0.109,0.416,0.838,1,0.108,0.866,0.932(>0.05).SHOX gene expression of CNE–4,CNE2,CNE3, CNE4,CNE5,CNE6,CNE7,CNE8,CNE12,CNE13, CNE14,CNE15areidentical to non transfection group.3.CNE-5,CNE-3,CNE-2,CNE9,CNE10,CNE11compare with nontransfection group,and obtain that the P are <.0001,<.0001,<.0001,0.015,0.01,0.007(<0.05).SHOX gene expression of CNE-5,CNE-3,CNE-2,CNE9,CNE10,CNE11are different from non transfection group.AndSHOX gene expression of these groups is higher than non transfectiongroup.In this assay,we found six enhancers to be active in the HEK293cells.