LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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fluorescent tag. (B) PCR amplification of genomic DNA purified from HHL-17 cells stably transduced with lentiviruses expressing YFP-K18. (C) Expression of K18-YFP in stable cell clones generated in HHL-17 cells (YFP is pseudo-colored in green). (Original magnification 630X with 2X optical zoom). Scale bars are shown in each panel. 4.1.5 The bi-cistronic lentiviral vectors can be used for live cell imaging experiments. To determine if these bi-cistronic lentiviral vectors could be used for studying cellular processes in live cells, the ability of Keratin 8 (K8) fused to CFP and K18-YFP to form an intermediate filament network in HEK-293 cells was determined. HEK-293 cells do not express K8 and K18, and thus were an ideal system to study filament formation. Lentiviral vectors expressing K18-YFP and K8-CFP downstream of the CMV promoter and the Ubc promoter respectively were generated. HEK 293 cells were transduced with lentiviruses expressing either K18-YFP or K8-CFP alone or in combination and visualized by confocal microscopy. HEK-293 cells transduced with either K18-YFP or K8-CFP alone did not demonstrate filament formation and instead showed the presence of distinct punctuate structures in the cytoplasm (Figure 4.1.6 a). However HEK-293 cells transduced with both K18-YFP and K8-CFP demonstrated filament network formation (Figure 4.1.6 a). These results are consistent with previously published work suggesting that keratin filaments are formed due to heterotypic interactions between a type I and type II keratin and that lack of one type results in the presence of cytoplasmic aggregates of the other member of the pair (98, 147, 150, 189, 238, 242, 320). Further, in HHL-17 cells, ectopic expression of both K18-YFP and K8-CFP results in the formation of a keratin filament network (figure 4.1.6 b). These results suggest that bi-cistronic lentiviral vectors can be used to co-express different fluorescent proteins for studying protein organization into higher order structures and protein colocalization. 102
Figure 4.1.6: Application of bi-cistronic lentiviral vectors in live cell imaging. (A) HEK 293 cells were transduced with either pLV-CMV- K18-YFP -IRES-Puro (Red pseudo color) or pLV-Ubc-K8-CFP-IRES-Puro (green pseudo color) or both constructs. No signal was observed in the YFP channel upon only K8-CFP transduction and no signal 103
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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
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1.2.2 The G1/S DNA Damage checkpoin
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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 and 98: Figure 4.1.1 Generation of lentivir
Page 99 and 100: 4.1.2 Generation of lentiviral vect
Page 101: transduction with the virus. Untran
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
Page 139 and 140: A B 139
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
Page 147 and 148: the desmosomal proteins recruitment
Page 149 and 150: Figure 4.3.25: Generation of stable
Page 151 and 152: 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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