The Study Of Early Growth Response Gene-1 In The Development Of Myopia In Mice

Chapter one:Construction and identification of plasmid mediated short hairpin RNA interference targeting the Egr-1 gene of miceObjective To construct and screen high-efficiency plasmid mediated short hairpin RNA interference (shRNA) targeting the early growth response factor-1 (Egr-1) of mice.Methods Based on the Egr-1 gene mRNA sequence of mouse (NM007913), we designed 3 hairpin oligonucleotide sequences, synthesized Oligo DNA of target sequences, annealed to form double-stranded DNA. By Age I and EcoR I digested the pGCSIL-GFP vector to generate recombinant plasmids containing shRNA 1#,2# and 3#, PCR screened clones and sequenced. Designed and constructed not for any specific gene plasmid as a negative control, named NC. The three shRNA expression plasmids and NC plasmid were co-transfected to HEK293 cells. Cells without any treatment have as blank control. Through observed the green fluorescent protein (GFP) expression and detected Egr-1 gene expression of mRNA and protein in cells by real-time fluorescence quantitative polymerase chain reaction (RQ-PCR) and western blotting, we identified the inhibitory efficiency of shRNA expression plasmid tageting Egr-1 gene. Groups were analyzed using ANOVA, between 3 sequences of experimental group were compared with q-test. Results The RNA interference sequences for Egr-1 gene of mice:1 #,2# and 3#, the plasmid of 1# sequence significantly inhibited the mRNA and protein expression of Egr-1 in cells (F mRNA=118.819, P=0.000; Fprotein=71.605, P=0.000).Conclusion We successfully constructed the efficient shRNA expression plasmid targeting murine Egr-1 gene.Chapter two:Construction the lentivirus vector mediated short hairpin RNA interference targeting the Egr-1 gene of mice and transfection to the retina of miceObjective To construct the lentivirus vector containing green fluorescent protein (GFP) and short hairpin RNA interference (shRNA) targeting the early growth response factor-1 (Egr-1) of mice and transfect to murine retina, exploring the appropriate method for drug delivery.Methods shRNA plasmid which is most efficient in inhibiting Egr-1 expression was named LV-shRNA (Egr-1). The LV-shRNA (Egr-1) recombinant plasmid and pHelper1.0, pHelper2.0 what the auxiliary plasmids of packaging of lentivirus were co-transfected to 293T cells, packaging and produce vector, diluted by the gradient, according to green fluorescent protein (GFP) positive cells under fluorescence microscope to calculate the virus titer. The packaged lentivirus vector was separately transfected to retina of C57BL/6 mice through intravitreal injection and subretina injection. Eyes of mice were enucleated for histological analysis GFP expression in retina was observed under fluorescence microscopy.Results①We successfully constructed the lentivirus vector containing GFP and shRNA targeting the Egr-1 gene of mice, method of dilution holes for the virus titer was 4×108 TU/ml;②Using the lentivirus vector system transfect retina of mice, we found GFP widely distributed in all retina layers including the retina pigment epithelium (RPE) cell layer after intravitreal injection; GFP restricted expressed in the outer retina after subretina injection.Conclusion We successfully constructed the lentivirus vector containing GFP and shRNA targeting the Egr-1 gene of mice, compared with the two methods of drug delivery, intravitreal injection was more efficient in transfection and wide distribution, provided a basis for experiments in vivo.Chapter three:The regulation effect of Egr-1 gene in mice myopiaObjective Have the intravitreal injection of lentivirus vector containing shRNA targeting the Egr-1 gene, observed the changes of refraction and axial length of mice, clarify the regulation of Egr-1 gene in the development of myopia in mice. Methods 180 15-days-old C57BL/6 mice were randomly divided into 3 groups:experimental group, negative control group and blank control group. The experimental group of mice right eyes had injected the lentivirus vector of LV-shRNA (Egr-1) which had preliminary study; negative control group of mice right eyes have injected the negative control lentivirus vector of LV-NC; without any treatment mice as blank control group. After 1,2 and 3 weeks of experiment, we measured the diopter of right eye of mice in each group, then killed mice after anesthesia, measured axial length of right eye of mice in each group; producted frozen sections, observe the GFP expression in the retina under the microscope to determine transfection efficiency; through using real-time fluorescence quantitative polymerase chain reaction (RQ-PCR), western blotting and immunofluorescence, we detect the expression of Egr-1 gene in murine retina; we observed retina changes under the microscope. Groups were analyzed using ANOVA, between 3 groups were compared with q-test..Results①Though RQ-PCR, western blotting and immunofluorescence, we found that the expression of Egr-1 was significantly reduced, in which experimental group injected eyes, especially the expression of Egr-1 have down-regulation the most significant after 1 week of experiment (F mRNA= 184.383, P= 0.000; F protein= 170.470, P= 0.000);②After 1 week of experiment, expression of GFP in murine retina was found in the experimental group and the negative control group after the injection of the lentivirus vector. After 2 weeks of experiment, the fluorescence intensity began to decay. After 3 weeks of experiment, expression of GFP in retina was weak;③After 1 week of experiment, (F diopter= 157.793,P=0.000; F axial length= 10.005, P=0.000) and after 2 weeks of experiment (Fdiopter= 182.603, P= 0.000; F axial length= 5.273, P= 0.007), the mice in experimental group which the injected eyes has a significant development of myopia, and with obvious extension of axial length; but After 3 weeks of experiment (F diopter= 1.259, P= 0.290; F axial length= 1.004, P= 0.371) the mice in experimental group which the injected eyes compare with the negative control group which injected eyes and control eyes, the refraction and axial length has no significant differences; negative control group which injected eyes at all stages of the experiment compare with the blank control eyes, the refraction and axial length has no significant difference (p> 0.05)④After 1 week of experiment, the most obvious trend of development of myopia in mice, take sections of this week and have HE staining, showed no significant morphological changes in the retina among 3 groups.Conclusion Through transfected the lentivirus vector containing shRNA targeting the Egr-1 gene to murine retina, the Egr-1 gene in mice retina have reduced, the refractive error and axial length tune the trend of myopia, confirmed that Egr-1 gene play an important role in the development of myopia in mice. We also found that it was safe and effective for lentivirus to transfected murine retina. The results provide the direction of thinking for gene therapy in myopia in the future.