LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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Chromosome Accession Number Number of mice Identity of mice number showing showing integrations integrations 1 NT_39170.7 3 6 &,12 2 NW_001030712.1 9 11,12,33,35,45 &51 NW_001030694.1 NW_001030686.1 3 NT_039207 2 38 NT_162143 4 NW_001030747 2 49 & 54 6 NW_039353 1 33 8 NT_078575.6 3 11 & 45 13 NT_039578 2 6 & 38 14 NW001030543 2 42 & 37 18 NW_001030631 NW_001030635.1 3 12 & 52 Table 4.2.2 Summary of the integration events observed in the different transgenic animals. The chromosome number is shown in column one while the number of the individual mice with an integration in these chromosomes is shown in column 4. The integration events in each chromosome are shown in the third column of this table with the appropriate accession number in column 2. As can be seen from the table, multiple mice show different combinations of integration events suggesting that transgene expression is independent of integration site. The number of assigned to the mice in this table is similar to the number reported in figure 3.9. 114
4.3 Generation of 14-3-3γ knockdown mice. The transition of cells from G 2 to M phase requires an active cdk1/cyclin B complex (345), which is activated by dephosphorylation of T14 and Y15 in cdk1 by the dual specificity phosphatase, cdc25C (103, 112). Cd25C itself is negatively regulated by phosphorylation on S216 during interphase and is not phosphorylated on S216 during mitosis (281). This site is a target for phosphorylation by DNA damage checkpoint kinases such as Chk1 and Chk2 (228, 281, 311). The phosphorylation of cdc25C on S216 creates a binding site for 14-3-3 proteins leading to the hypothesis that 14-3-3 proteins negatively regulate cdc25C function (69, 281). Binding of 14-3-3 proteins to cdc25C leads to its retention in the cytoplasm thus preventing mitotic progression (69, 126). Mutation of Serine 216 to Alanine (S216A), results in loss of 14-3-3 binding and a pancellular localization of cdc25C (69, 126). These results suggest that 14-3-3 proteins regulate cdc25C function in part by restricting it to the cytoplasm in interphase. Further, it has been reported that 14-3-3 proteins prevent interaction between cdc25C and its substrate, the cyclin B/cdk1 complex (244). Therefore, 14-3-3 proteins regulate cdc25C function at multiple levels. It has been demonstrated that 14-3-3 β, ε, γ, σ and ζ bind to wild type cdc25C in vitro but not to the S216A mutant (71). However, coimmunoprecipitation experiments revealed that only 14-3-3ε and 14-3-3γ form a complex with and inhibit cdc25C function in vivo (71). Similarly, cells lacking either 14-3-3ε or 14-3-3γ show an override of the S phase and DNA damage checkpoints and these are due to the inability of these cells to regulate cdc25C function (157, 354). Further, a structural motif conserved in 14-3-3ε and 14-3-3γ mediates specific complex formation with cdc25C in vivo (354). These results suggest that the activity of different 14-3-3 isoforms is not redundant and that each 14-3-3 protein may selectively bind to different ligandssuggesting that loss of 14-3-3ε and 14-3-3γ leads to defects in checkpoint function (157, 354). Given these results, we wished to determine whether loss of 14-3-3ε and 14-3-3γ in the mouse would affect growth and development. 115
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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
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cytoplasmic accumulation of cdc25 p
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14-3-3 binding site. 14-3-3 binding
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G2 arrest and induced premature chr
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emoval of S216 phosphorylation as i
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embryogenesis, a loss of cdc25A can
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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 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: sequenced and the distribution amon
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
Page 153 and 154: compared to vector control cells. (
Page 155 and 156: 4.4 Generation of 14-3-3ε knockdow
Page 157 and 158: into the interstitial spaces of the
Page 159 and 160: Figure 4.4.3: 14-3-3ε knockdown in
Page 161 and 162: Figure 4.4.4: Patchy hair loss in 1
Page 163 and 164: 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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