LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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form pBSK-F2A8 was further sub cloned in pLKO.1 MCS to generate pLKO.1 EGFP-f Puro which expresses both EGFP-f and a shRNA downstream of the U6 promoter (figure 4.1.2 g). Figure 4.1.2 Generation of pBSK-F2 shuttle vector (A) Schematic representation of pBSK-F2. (B) Transcription factor binding site in EF1 promoter. (C) EGFP-f reporter assay in HEK 293 cells using pBSK-F2 and pBSK-F2A8 shuttle vector as determined by Flow cytometry. (D) Graphical representation of percentage of GFP positive as obtained in EGFP-f reporter assay. (E) Westren blot analysis for the expression of EGFP-f in HEK 293 cells transfected with pBSK-F2 or pBSK-F2A8 shuttle vector using GFP monoclonal antibody, actin was probed for loading control. (F) Confocal image of HEK 293 cells expressing EGFP-f. (G) Schematic representation of pLKO EGFP-f Puro. 98
4.1.2 Generation of lentiviral vectors for cDNA expression. The lentiviral vector backbone for the expression of cDNA was derived from pLKO.1 puro (343). An expression cassette containing the elongation factor1α (EF1-α) promoter driving the expression of EGFP-f, followed by SV40 poly-adenylation sequence in pBSK-F2A8 was modified by replacing EGFP-f with an MCS containing unique restriction sites to generate pBSK-FM3 (figure 4.1.3 a & b). Further the expression cassette from pBSK-FM3 was subcloned in pLKO.1 puro using ClaI to generate pEF.55 lentiviral vector (figure 4.1.3 c). Figure 4.1.3 Generation of lentiviral vectors for the expression of cDNA (A) Schematic representation of pBSK-FM3. (b)Multiple cloning sites depicting the restriction sites introduced in pBSK-FM3. (c) Schematic representation of pEF55 lentiviral vector. 4.1.3 Generation of bi-cistronic lentiviral vectors. The lentiviral vector backbone for the expression vectors was derived from the pTRIPZ vector (Open Biosystems). The CMV promoter was cloned into the pTRIPZ vector as described in Materials and Methods followed by the introduction of a MCS upstream of various fluorescent proteins (EGFP, ECFP, EYFP, mCherry and dsRed) and the IRES PuroR cassette to obtain the resultant bi-cistronic lentiviral vector pLV- FP-IRES-Puro (figure 4.1.4 a). A notable shortcoming of several of the currently available bi-cistronic 99
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Generation of knockdown mice that l
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STATEMENT BY AUTHOR This dissertati
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CERTIFICATE I certify that the thes
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I am thankful to Mr Uday Dandekar f
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3.16 Isolation of Genomic DNA (gDNA
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A synopsis of thesis to be submitte
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esults in loss of 14-3-3 binding an
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and NheI (NEB) to remove tetO-CMV,
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Hanging drop and wound healing assa
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360). To generate transgenic animal
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asement of the seminiferous tubules
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normal female, pups were screened f
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14-3-3ε only in the presence of HP
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proteins are expressed in testis (3
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5. Lalit Sehgal, Srikanth B., Khyat
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Presented a poster at 33rd All Indi
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shRNA siRNA TBS-T U.V WT C IF DSG D
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List of Figures: Page No 1. Figure
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37. Figure 4.3.20: 14-3-3γ interac
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CHAPTER 1 INTRODUCTION 39
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1.1.1 Cyclin Cdk complexes Eukaryot
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1.1.1.c Cyclin A cdk complexes The
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of which are associated with acquis
Page 47 and 48: 1.2.2 The G1/S DNA Damage checkpoin
Page 49 and 50: complexes. DNA damage induced degra
Page 51 and 52: cytoplasmic accumulation of cdc25 p
Page 53 and 54: 14-3-3 binding site. 14-3-3 binding
Page 55 and 56: G2 arrest and induced premature chr
Page 57 and 58: emoval of S216 phosphorylation as i
Page 59 and 60: embryogenesis, a loss of cdc25A can
Page 61 and 62: dimer was essential for Raf activat
Page 63 and 64: of enhanced apoptosis of peripheral
Page 65 and 66: the generation of transgenic mice a
Page 67 and 68: CHAPTER 2 AIMS AND OBJECTIVES 67
Page 69 and 70: CHAPTER 3 MATERIALS AND METHODS 69
Page 71 and 72: Name of oligonucleotide EF1α Forwa
Page 73 and 74: cleavage sites GAAGGTATATTGCTGTTGAC
Page 75 and 76: R E1 A GAACGAATAGTGAAGCCACAGATGTA s
Page 77 and 78: 100mm 25ug 225ul 250ul 500 1000ul T
Page 79 and 80: for the transgene were considered a
Page 81 and 82: solution in TBS-T containing 0.02%
Page 83 and 84: mounting agent. Confocal images wer
Page 85 and 86: 3.26 Antibodies used for Western bl
Page 87 and 88: each plate was added cold 500ml of
Page 89 and 90: 3.32 Hanging drop assay to determin
Page 91 and 92: 3.37 Biodistribution of 18 F -FDG u
Page 93 and 94: 2.5M NaCl 150mM 60ml Tween-20 (Sigm
Page 95 and 96: CHAPTER 4 RESULTS 95
Page 97: Figure 4.1.1 Generation of lentivir
Page 101 and 102: transduction with the virus. Untran
Page 103 and 104: Figure 4.1.6: Application of bi-cis
Page 105 and 106: Figure 4.1.7: Application of bi-cis
Page 107 and 108: Figure 4.2.1: Infection of morulae
Page 109 and 110: Figure 4.2.2: Generation of transge
Page 111 and 112: Figure 4.2.3: EGFP-f expression in
Page 113 and 114: sequenced and the distribution amon
Page 115 and 116: 4.3 Generation of 14-3-3γ knockdow
Page 117 and 118: numbering). Alignment was done usin
Page 119 and 120: presence of EGFP-f by performing po
Page 121 and 122: the mouse tested infected with vect
Page 123 and 124: Figure 4.3.7: Staging of 14-3-3γ k
Page 125 and 126: containing spermatozoa is on the Y-
Page 127 and 128: Figure 4.3.9: 14-3-3γ knockdown le
Page 129 and 130: Figure 4.3.11: 14-3-3γ knockdown i
Page 131 and 132: intercellular space (12, 110, 115,
Page 133 and 134: Figure 4.3.13: 14-3-3γ loss leads
Page 135 and 136: Figure 4.3.16: Localization of PKP3
Page 137 and 138: Figure 4.3.18: Reintroduction of sh
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Page 141 and 142: from our laboratory demonstrated th
Page 143 and 144: Figure 4.3.21: Plakoglobin recruitm
Page 145 and 146: 4.3.23 (A and B)). These proteins g
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Figure 4.3.25: Generation of stable
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KIF5B, however its reduction does n
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compared to vector control cells. (
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4.4 Generation of 14-3-3ε knockdow
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into the interstitial spaces of the
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Figure 4.4.3: 14-3-3ε knockdown in
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Figure 4.4.4: Patchy hair loss in 1
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follicle formation marked by black
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Figure 4.4.7: Phenotypes in 14-3-3
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compared to WT lungs (indicated by
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compared to wild type (Figure 4.4.1
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and CD4 positive lyumphocyte number
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Figure 4.4.13: Bone marrow cells fr
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Figure 4.4.15: Generation of stable
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Figure 4.4.16: NIH3T3 cells lacking
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Figure 4.4.18: Generation of induci
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CHAPTER 5 DISCUSSION 181
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to generate transgenic animals expr
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transgenic research and the use of
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(reviewed in (152, 374)). Kinesins
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subtypes known to date. Type IV col
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Figure 5.1 Schematic representation
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loss of 14-3-3ε the T cell activat
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BIBLIOGRAPHY: 1. Abraham, R. T. 200
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29. Blomer, U., L. Naldini, T. Kafr
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58. Cheng, C. Y., and D. D. Mruk. 2
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90. Elia, A. E., P. Rellos, L. F. H
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118. Girard, F., U. Strausfeld, J.
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150. Herrmann, H., M. Haner, M. Bre
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and activation of a cyclin E-cdk2 c
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213. Liu, D., J. Bienkowska, C. Pet
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242. Moll, R., W. W. Franke, D. L.
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274. Pagano, M., R. Pepperkok, F. V
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305. Russell, L. D. 1978. The blood
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inhibitor of metalloproteases-1 in
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364. Tsunekawa, N., M. Matsumoto, S
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397. Yashiro, M., N. Nishioka, and
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Research: Biology to Prevention to
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LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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