LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

More documents

Recommendations

Info

cell lines and could be used for live cell imaging techniques such as FRET (manuscript submitted). Development of a new technique for the generation of transgenic animals. The generation of genetically modified mice has spurred great advances in our understanding of various aspects of growth and development. Multiple technologies have used either injection into a one celled embryo followed by implantation into a pseudo pregnant mother (121), or used stem cell aggregation techniques to generate either knockout (123) or knockdown mice (360). These experiments are expensive, labor-intensive, timeconsuming and require several female donors. Spermatogonial stem cells are responsible for the production of spermatozoa (74) and are an appropriate target for germline modification (254). Nagano et. al. have generated transgenic mice by infecting spermatogonial stem cells in vitro with recombinant retroviruses followed by xenogenic transplantation of the cells into the testes of a male mouse (253). In some cases the recipient mice were unreceptive to the donor spermatogonial cells (253) and the overall success rate was rather low. Similarly, in vivo transduction of testicular germ cells with retroviral constructs carrying a lacZ gene, resulted in a poor success rate of 2.8% (169). The low success rates post implantation, however precluded these from replacing embryonic injection. Similar experiments using lentiviruses, resulted in better success rates (138). Recombinant lentiviruses expressing EGFP-f (EGFP tagged with a farnesylation signal) were injected into the intertubular spaces of the testis targeting undifferentiated spermatogonia present in the seminiferous tubules. Injection into the intertubular space allows the lentivirus to infect undifferentiated spermatogonial cells located at the 20
asement of the seminiferous tubules (168). Five to ten microlitres of recombinant EGFPf expressing lentiviruses (5 x 10 6 TU/ml) was injected into the intertubular spaces of the testis of 28 day old Crl:CFW(SW) male mice. These male mice, referred to as pre-founder mice, were mated with wild type females of the same strain 35 days post infection. Transgenic pups were generated from mating experiments with three independently derived pre-founder mice at an overall rate greater than 60%, a rate that is much higher than previously reported with conventional transgenic protocols or with retroviral infection of spermatogonial stem cells in vitro or in vivo (169, 253). The experiments resulted in the development of a simple, cost effective, and efficient technique for the generation of transgenic mice by in vivo transduction of the desired gene/shRNA construct into undifferentiated spermatogonia of the testis (322). This technology does not compromise the fertility of the off-spring, resulting in germline transmission of the transgene, using a limited number of animals . Generation of mice with a knockdown of 14-3-3ε or 14-3-3γ. To generate knockdown mice for 14-3-3γ, lentiviruses that express EGFP-f and shRNA’s that target 14-3-3γ were injected into the efferent duct of the developing testes to infect spermatogonial stem cells. None of the mice injected with lentiviruses expressing shRNA’s targeting 14-3-3γ produced pups when mated with wild type female mice. Even pups that lack the transgene were not produced in these mating experiments suggesting that male mice injected with the 14-3-3γ knockdown viruses were sterile. To determine if the mice infected with the 14-3-3γ knockdown constructs were sterile, three male mice injected with viruses encoding the 14-3-3γ shRNA were sacrificed and a histochemical analysis performed on sections from the testes. Wild type mice showed the presence of multiple spermatids at 21
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 and 18: Hanging drop and wound healing assa
Page 19: 360). To generate transgenic animal
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
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 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
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
Page 165 and 166:
Figure 4.4.7: Phenotypes in 14-3-3
Page 167 and 168:
compared to WT lungs (indicated by
Page 169 and 170:
compared to wild type (Figure 4.4.1
Page 171 and 172:
and CD4 positive lyumphocyte number
Page 173 and 174:
Figure 4.4.13: Bone marrow cells fr
Page 175 and 176:
Figure 4.4.15: Generation of stable
Page 177 and 178:
Figure 4.4.16: NIH3T3 cells lacking
Page 179 and 180:
Figure 4.4.18: Generation of induci
Page 181 and 182:
CHAPTER 5 DISCUSSION 181
Page 183 and 184:
to generate transgenic animals expr
Page 185 and 186:
transgenic research and the use of
Page 187 and 188:
(reviewed in (152, 374)). Kinesins
Page 189 and 190:
subtypes known to date. Type IV col
Page 191 and 192:
Figure 5.1 Schematic representation
Page 193 and 194:
loss of 14-3-3ε the T cell activat
Page 195 and 196:
BIBLIOGRAPHY: 1. Abraham, R. T. 200
Page 197 and 198:
29. Blomer, U., L. Naldini, T. Kafr
Page 199 and 200:
58. Cheng, C. Y., and D. D. Mruk. 2
Page 201 and 202:
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
Page 207 and 208:
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
Page 215 and 216:
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
show all

LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?