LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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(mm 3 ) was calculated by the formula ½ LV 2 where L is the largest dimension and V its perpendicular dimension, as previously reported (292, 389). RESULTS To determine if loss of 14-3-3 and/or 14-3-3 in MESC results in mislocalization of cdc25C. To address whether the loss of 14-3-3 or 14-3-3 results in mislocalization of cdc25C, we first determined whether 14-3-3 and 14-3-3formed a complex with mouse cdc25C by performing GST pull down assays. While both 14-3-3 and 14-3-3formed a complex with human cdc25C in this assay, neither protein could form a detectable complex with mouse cdc25C. In humans, 14-3-3 proteins bind to cdc25C in vitro and in vivo upon phosphorylation at a Serine residue at position 216; however a comparison of sequences of cdc25C proteins from different species demonstrated that this sequence was absent in mouse, rat and hamster cdc25C but was present in cdc25C from Xenopus laevis and Sus domestica. These results lead us to conclude that 14-3-3 proteins might regulate cell cycle progression in a cdc25C independent fashion in the mouse. Generation of constitutive and inducible lentiviral vectors for transgene expression or the generation of knockdown mice. Within the last decade, multiple lentiviral vectors have been developed as tools for gene delivery and for therapeutic and experimenta l transgenic applications (66, 105, 173, 226, 234, 284, 285, 295, 336, 400) as they permit in vivo gene delivery to terminally differentiated cells in multiple tissue types (66, 105, 173, 183). Because of their ability to transduce quiescent cells, lentiviral vectors are also promising tools for ex vivo genetic modification of stem cells (29, 297), which reside almost exclusively in the G0/G1 phase of the cell cycle and can also be used to generate transgenic mouse models of human disease (3, 138, 169, 226, 253, 255, 256, 284, 307, 18
360). To generate transgenic animals using lentiviral vectors, we first generated lentiviral vectors capable of expressing both a shRNA targeting the gene of interest and EGFP-f permitting identification of transgenic cells in vitro and in vivo. Previous results had indicated that a lentiviral vector in which cDNA’s were downstream of the CMV promoter was not effective in achieving lentiviral transduction and expression in germ line stem cells (256). Therefore an expression cassette containing the elongation factor 1a (EF1-α) promoter driving the expression of EGFP-f , followed by the SV40 polyadenylation sequence was assembled in pBSK. The assembled cassette was further sub cloned in pLKO.1 (343) to generate a vector that could express EGFP-f from the EF1-α promoter and an shRNA downstream of the U6 promoter. The ability of the lentiviral vector to express EGFP-f was determined by infecting HEK 293 cells with recombinant lentiviral particles and detecting the presence of EGFP-f using a combination of immunofluorescence microscopy, Western blotting and flow cytometry. A lentiviral vector PS-18 expressing shRNA under U6 promoter was also generated (122). Similarly, HIV-1 based self inactivating bi-cistronic lentiviral vector systems were generated using either the human cytomegalovirus immediate early promoter (CMV) or the Ubiquitin C (Ubc) promoter upstream of a unique multiple cloning site (MCS). Different flourescent proteins (EGFP, mCherry, ECFP, EYFP and dsRed) have been introduced downstream of the MCS permitting the generation of multiple differentially tagged fusion constructs. An encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), positioned between the fluorescent protein and a gene conferring resistance to Puromycin (PuroR), facilitates cap-independent translation of PuroR from an internal start site at the IRES/Puro R junction. These lentiviral vectors were efficient in transducing various target 19
Page 1 and 2: Generation of knockdown mice that l
Page 3 and 4: STATEMENT BY AUTHOR This dissertati
Page 5 and 6: CERTIFICATE I certify that the thes
Page 7 and 8: I am thankful to Mr Uday Dandekar f
Page 9 and 10: 3.16 Isolation of Genomic DNA (gDNA
Page 11 and 12: A synopsis of thesis to be submitte
Page 13 and 14: esults in loss of 14-3-3 binding an
Page 15 and 16: and NheI (NEB) to remove tetO-CMV,
Page 17: Hanging drop and wound healing assa
Page 21 and 22: asement of the seminiferous tubules
Page 23 and 24: normal female, pups were screened f
Page 25 and 26: 14-3-3ε only in the presence of HP
Page 27 and 28: proteins are expressed in testis (3
Page 29 and 30: 5. Lalit Sehgal, Srikanth B., Khyat
Page 31 and 32: Presented a poster at 33rd All Indi
Page 33 and 34: shRNA siRNA TBS-T U.V WT C IF DSG D
Page 35 and 36: List of Figures: Page No 1. Figure
Page 37 and 38: 37. Figure 4.3.20: 14-3-3γ interac
Page 39 and 40: CHAPTER 1 INTRODUCTION 39
Page 41 and 42: 1.1.1 Cyclin Cdk complexes Eukaryot
Page 43 and 44: 1.1.1.c Cyclin A cdk complexes The
Page 45 and 46: 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
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CHAPTER 3 MATERIALS AND METHODS 69
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Name of oligonucleotide EF1α Forwa
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cleavage sites GAAGGTATATTGCTGTTGAC
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R E1 A GAACGAATAGTGAAGCCACAGATGTA s
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100mm 25ug 225ul 250ul 500 1000ul T
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for the transgene were considered a
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solution in TBS-T containing 0.02%
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mounting agent. Confocal images wer
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3.26 Antibodies used for Western bl
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each plate was added cold 500ml of
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3.32 Hanging drop assay to determin
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3.37 Biodistribution of 18 F -FDG u
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2.5M NaCl 150mM 60ml Tween-20 (Sigm
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CHAPTER 4 RESULTS 95
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Figure 4.1.1 Generation of lentivir
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4.1.2 Generation of lentiviral vect
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transduction with the virus. Untran
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Figure 4.1.6: Application of bi-cis
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Figure 4.1.7: Application of bi-cis
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Figure 4.2.1: Infection of morulae
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Figure 4.2.2: Generation of transge
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Figure 4.2.3: EGFP-f expression in
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sequenced and the distribution amon
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4.3 Generation of 14-3-3γ knockdow
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numbering). Alignment was done usin
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presence of EGFP-f by performing po
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the mouse tested infected with vect
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Figure 4.3.7: Staging of 14-3-3γ k
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containing spermatozoa is on the Y-
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Figure 4.3.9: 14-3-3γ knockdown le
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Figure 4.3.11: 14-3-3γ knockdown i
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intercellular space (12, 110, 115,
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Figure 4.3.13: 14-3-3γ loss leads
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Figure 4.3.16: Localization of PKP3
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Figure 4.3.18: Reintroduction of sh
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A B 139
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from our laboratory demonstrated th
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Figure 4.3.21: Plakoglobin recruitm
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4.3.23 (A and B)). These proteins g
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the desmosomal proteins recruitment
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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.
Page 205 and 206:
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
Page 219 and 220:
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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