Efficiency of targeted interference on early growth response factor-1 by lentivirus vector containing shRNA transfected into mouse retina

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Author:
JIANG Jing-jing(Department of Ophthalmology,Xiangya Hospital,Central South University,Changsha 410008,China)
LIU Shuang-zhen(Department of Ophthalmology,Xiangya Hospital,Central South University,Changsha 410008,China)
WEN Dan(Department of Ophthalmology,Xiangya Hospital,Central South University,Changsha 410008,China)
WANG Sha(Department of Ophthalmology,Xiangya Hospital,Central South University,Changsha 410008,China)
Journal Title:
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
Issue:
Volume 17, Issue 02, 2011
DOI:
10.3760/cma.j.issn.1674-1927.2011.02.004
Key Word:
Early growth response factor-1;RNA interference;Transfection;Lentivirus vector;Gene expression regulation

Abstract: Objective To construct the lentivirus vector containing green fluorescent protein (GFP)and early growth response factor-1 short hairpin RNA(Egr-1 shRNA),and to determine the efficiency of interference on Egr-1 gene in lentivirus vector-transfected mouse retinal tissue.Methods Based on the target sequences of Egr-1 shRNA screened and identified previously,the LV-shRNA(Egr-1)containing GFP and Egr-1 shRNA Was constructed.Twenty 15-day-old C57BL/6 mice were randomly divided into experimental group and negative control group(n=10 each).LV-shRNA(Egr-1)lentivirus vector Was transfected into the right eyes in the experimental group with intravitreal injection.LV-NC lentivirus vector that did not target at any specific gene Was also transfected into the right eyes in the negative control group,while the left eyes were untreated in both two groups as the blank control group.After 2 weeks,the transfection of lentiviral vector was examined under fluorescence microscope.Moreover,real-time quantitative polymerase chain reaction (RQ-PCR), Western blot and immunofluorescence were used to detect the expression of Egr-1 gene for evaluation of the interference efficiency. Results After lentiviral vector was intravitreally injected and transfected into the mouse retinal tissue, GFP was found to be widely distributed in the whole layer of the retina including the retinal pigment epithelium. RQPCR showed significantly down-regulated expression of Egr-1 mRNA in the injected eyes of experimental group compared with blank or negative controls (0.290±0.074 vs 1.006±0.033, 1.010± 0.086, all P<0.001) , with the inhibition rate being 71.29%. Western blot demonstrated that the expression of Egr-1 protein was remarkably down-regulated in the injected eyes of experimental group (0.224±0.035 vs 0.674±0.050, 0.688±0.049, all P<0.00l) , with the inhibition rate being 67.44%. Furthermore, immunofluorescence revealed no expression of fluorescence protein, except sparse distribution of Egr-1 positive cells in the inner nuclear layer of injected eyes of the experimental group. Conclusions The lentivirus vector containing GFP and Egr-1 shRNA is successfully constructed, and it may have high transfection efficiency and a wide distribution in the intravitreally injected mice eyes. Furthermore, the lentivirus vector has a high inhibition efficiency of Egr-1 gene in the mouse retina.

  • [1]Hannon GJ.RNA interference.Nature,2002,418:244-251.
  • [2]Kissler S,Van Parijs L.Exploring the genetic basis of disease using RNA interference.Expert Rev Mol Diagn,2004,4:645-651.
  • [3]Rao DD,Senzer N,Cleary MA,et al.Comparative assessment of siRNA and shRNA off target effects:what is slowing clinical development.Cancer Gene Ther,2009,16:807-809.
  • [4]Bassik MC,Lebbink RJ,Churchman LS,et al.Rapid creation and quantitative monitoring of high coverage shRNA libraries.Nat Methods,2009,6:443-445.
  • [5]Chen SJ,Ning H,Ishida W,et al.The early-immediate gene EGR1 is induced by transforming growth factor-beta and mediates stimulation of collagen gene expression.J Biol Chem,2006,281:21183-21197.
  • [6]Zeng XR,Sun Y,Wenger L,et al.Basic calcium phosphate crystalinduced Egr-1 expression stimulates mitogenesis in human fibroblasts.Biochem Biophys Res Commun,2005,330:658-664.
  • [7]Shafarenko M,Liebermann DA,Hoffman B.Egr-1 abrogates the block imparted by c-Myc on terminal M1 myeloid differentiation.Blood,2005,106:871-878.
  • [8]Brand C,Schaeffel F,Feldkaemper MP.A microarray analysis of retinal transcripts that are controlled by image contrast in mice.Mol Vis,2007,13:920-932.
  • [9]Bitzer M,Schaeffel F.Defocus-induced changes in ZENK expression in the chicken retina.Invest Ophthalmol Vis Sci,2002,43:246-252.
  • [10]Zhong X,Ge J,Smith EL 3rd,et al.Image defocus modulates activity of bipolar and amacrine cells in macaque retina.Invest Ophthalmol Vis Sci,2004,45:2065-2074.
  • [11]Ashby R,McCarthy CS,Maleszka R,et al.A muscarinic cholinergic antagonist and a dopamine agonist rapidly increase ZENK mRNA expression in the form-deprived chicken retina.Exp Eye Res,2007,85:15-22.
  • [12]Schippert R,Burkhardt E,Feldkaemper M,et al.Relative axial myopia in Egr-1 (ZENK) knockout mice.Invest Ophthalmol Vis Sci,2007,48:11-17.
  • [13]Fuchs U,Borkhardt A.The application of siRNA technology to cancer biology discovery.Adv Cancer Res,2007,96:75-102.
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