LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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4.4 Generation of 14-3-3ε knockdown mice. 155 5. CHAPTER 5 : DISCUSSION 181 5.1 Generation of lentiviral vectors. 182 5.2 Novel protocol for the generation of transgenic mice. 183 5.3 Generation of knockdown mice for 14-3-3ε and 14-3-3γ. 185 6. BIOLIOGRAPHY 195 7. PUBLICATIONS 222 10
A synopsis of thesis to be submitted to the Homi Bhabha National Institute For the degree of Doctor of Philosophy In Life Sciences Name of the candidate: Lalit Sehgal Title of the thesis: Generation of knockdown mice that lack 14-3-3 and 14-3-3 using RNA interference Degree: Ph.D. Subject: Life Science Registration number: LIFE09200604002 Date of registration: 01/09/2006 Name and designation of the guide : Dr. Sorab N. Dalal S. O. ‘F’ ACTREC, Assistant Professor HBNI Institute: Tata Memorial Centre, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Kharghar, Navi Mumbai – 410210 11
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: 3.16 Isolation of Genomic DNA (gDNA
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 and 18: Hanging drop and wound healing assa
Page 19 and 20: 360). To generate transgenic animal
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
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dimer was essential for Raf activat
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of enhanced apoptosis of peripheral
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the generation of transgenic mice a
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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
Page 105 and 106:
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
Page 181 and 182:
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
Page 199 and 200:
58. Cheng, C. Y., and D. D. Mruk. 2
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90. Elia, A. E., P. Rellos, L. F. H
Page 203 and 204:
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
Page 209 and 210:
213. Liu, D., J. Bienkowska, C. Pet
Page 211 and 212:
242. Moll, R., W. W. Franke, D. L.
Page 213 and 214:
274. Pagano, M., R. Pepperkok, F. V
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305. Russell, L. D. 1978. The blood
Page 217 and 218:
inhibitor of metalloproteases-1 in
Page 219 and 220:
364. Tsunekawa, N., M. Matsumoto, S
Page 221 and 222:
397. Yashiro, M., N. Nishioka, and
Page 223:
Research: Biology to Prevention to
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LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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