Construction and characterisation of a stably transfected BHK cell line permanently secreting the canine interleukin 12 as a source for adoptive cancer immunotherapy in dogs [Elektronische Ressource] / by Vladimir Kocoski
92 Pages
English
Downloading requires you to have access to the YouScribe library
Learn all about the services we offer

Construction and characterisation of a stably transfected BHK cell line permanently secreting the canine interleukin 12 as a source for adoptive cancer immunotherapy in dogs [Elektronische Ressource] / by Vladimir Kocoski

Downloading requires you to have access to the YouScribe library
Learn all about the services we offer
92 Pages
English

Description

CONSTRUCTION AND CHARACHTERISATION OF A STABLY TRANSFECTED BHK CELL LINE PERMANENTLY SECRETING THE CANINE INTERLEUKIN 12 AS A SOURCE FOR ADOPTIVE CANCER IMMUNOTHERAPY IN DOGS Inaugural Dissertation submitted to the Faculty of Veterinary Medicine in partial fulfillment of the requirements for the PhD-Degree of the Faculties of Veterinary Medicine and Medicine of the Justus Liebig University Giessen by Kocoski, Vladimir of Struga, Republic of Macedonia Giessen 2008 From the Institute of Veterinary Pathology Director: Prof. Dr. Manfred Reinacher of the Faculty of Veterinary Medicine of the Justus Liebig University Giessen First Supervisor and Committee Member: Prof. Dr. Eberhard Burkhardt Second Supervisor and Committee Member: Prof. Dr. Lothar Stitz Committee Members: Prof. Dr. Dr. Hans Michael Piper Prof. Dr. Holger Hackstein Date of Doctoral Defence: 31.03.

Subjects

Informations

Published by
Published 01 January 2008
Reads 9
Language English
Document size 1 MB

Exrait

  
 
 
 
 
 
 
 
 
 
 
  
  
  
  
  
  
 
    CONSTRUCTION AND CHARACHTERISATION OF A STABLY TRANSFECTED BHK CELL LINE PERMANENTLY SECRETING THE CANINE INTERLEUKIN 12 AS A SOURCE FOR ADOPTIVE CANCER IMMUNOTHERAPY IN DOGS         Inaugural Dissertation submitted to the Faculty of Veterinary Medicine in partial fulfillment of the requirements for the PhD-Degree of the Faculties of Veterinary Medicine and Medicine of the Justus Liebig University Giessen          by Kocoski, Vladimir  of Struga, Republic of Macedonia    Giessen 2008         
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
                                                   
  
  
  
  
  
  
  
  
  
  
  
  
 
 
  
  
  
  
  
  
 
 
  
  
 
 
 
 
ulty
rs
        From the Institute of Veterinary Pathology Director: Prof. Dr. Manfred Reinacher Veterinary Medicine of the Justus Liebig Unive          
of
 
 
          
 
 
  
Da
te
 
nce
 of Doctoral Defe 31.03.2008    
 
:
 
 First Supervisor and Committee Member: Prof. Dr. Eberhard Burkhardt  Second Supervisor and Committee Member: Prof. Dr. Lothar Stitz  Committee Members:  Prof. Dr. Dr. Hans Michael Piper Prof. Dr. Holger Hackstein     
of
n
he
t
c
Fa
es
se
ity
Gi
                                                   
  
 
 
 
 
 
 
 
 
 
                I declare that I have completed this dissertation single-handedly without the unauthorized help of a second party and only with the assistance acknowledged therein. I have appropriately acknowledged and referenced all text passages that are derived literally from or are based on the content of published or unpublished work of others, and all information that relates to verbal communications. I have abided by the principles of good scientific conduct laid down in the charter of the Justus Liebig University of Giessen in carrying out the investigations described in the dissertation.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
              
                             
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TO
 
M
TS
EN
R
Y
PA
  
 
 
  
  
  
  
  
 
 
 
 
 
 
 
 
  
  
  
  
  
  
  
  
 
  
  
 
  
  
  
  
  
 
3.1.1.2 
3.1.1.1 
3.1.2 
3.1.1.3 
3.1.4 
3.1.3 
3.1.5 
3.1.6 
3.2.1 
3.2 
3.2.2 
3.2.3 
 14
 14
 12
 13
 11
 11
 10
 10
 
 
 
 
 
 
 
 
 
2.2 
2.2.1 
2.2.2 
 
 
2.4 
 
 
2.6 
2.6.1 
2.3 
2.5 
3.2.4 
2.6.2 
3.3.1 
3.3 
3 
3.1.1 
2.6.3 
3.1 
 
 
 
 
 
 
 
 
                                          
 
 
Protein expression in mammalian cells
 
 
 
General principles
 
The Tet System for inducible mammalian expression
Biology of IL-12 in human and mice 
INTRODUCTION  
LITERATURE OVERVIEW
 
 
 
DNA Cloning
 
Cloning
MATERIALS AND METHODS
Cell proliferation
Cytotoxicity assay
 
Taq Polymerase
KOD Hot Start DNA Polymerase
Polymerase Chain Reaction
IL-12 as a single chain
 
Biology of IL-12 in the dog
 
PBMC Isolation
MC
Investigation of the IL-12 effects on PB
 
 
 
 
 
Plasmid DNA extraction
Construction of the recombinant canine single chain IL-12 (rcscIL-12)
 
 
 
A-overhang addition and TA cloning
 
 
Ligation
Transformation of E.coli
Gel electrophoresis
Restriction digestion
 
 
 
 
 
 
 
 
 
 14
 14
 
 
 
 
 
 
 
 
 
 20
 
 22
 21
 22
 22
 3
 1
 4
 3
 5
 4
 
 
 6
 
 
 
 
 
 
 
 
 
 
 
 
 
2.1 
2 
1 
 Content
 
 
 
 
 
 
 
 
 
 
 
 
(DpnI site-directed mutagenesis)
 
Construction of the rcscIL-12
  
 19
Completion of the cDNA coding for the IL-12 p35 sequence
Correction of the IL-12 p40 cDNA sequence
 19
 
Establishing of a stably transfected BHK Tet-On cell line
Stable transfection
 
 
 
 
Construction of the rcscIL-12/IREs/Luciferase/pTRUE plasmid
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 18
 17
 17
 16
 16
 15
 15
6 
7 
4.3.2 
4.3.1 
4.2.3 
4.3 
4.2.2.1 
4.2.2 
4.2.1 
4.1.4 
4.2 
4.1.2 
4.1.1 
4 
4.1 
4.1.3 
3.4.4 
3.4.5 
 
3.4.3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 32
 
 
 
 33
 30
 32
 32
 57
 55
 50
 29
5 
4.3.4 
4.3.3 
3.4.2 
Investigation of the biological effects of rcscIL-12 on canine PBMC
 45
Resting canine PBMC do not produce IFN-γ 45after stimulation with IL-12
 34
 
 
 
 42
in IL-2 lymphoblasts
Proliferation of canine IL-2 lymphoblasts upon IL-12 stimulation
Evaluation of the cytotoxic activity of canine IL-2 lymphoblasts
 46
 34
IL-12 requires presence of IL-2 to induce IFN-γ ion tcudorp 
   
  
SUMMARY   
 
ZUSAMMENFASSUNG 
 
upon stimulation with IL-12
 47
 
DISCUSSION  
 
 48
 
 
 
 
 
 
Correction of the cDNA coding for the IL-12 p40 sequence
    
 
 
 
 
Repair of the cDNA coding for the IL-12 p35 sequence
 
 
 
 36
 
Establishing of rcscIL-12 containing BHK Tet-On cell line 
 
Construction of the rcscIL-12
Construction of the rcscIL-12/IRES/Luciferase/pTRUE plasmid
 
 
 3 6
Transfection of the BHK Tet-On cell line
 
 
 
 
Confirmation of bioactivity of the IL-12-
 41
 
 
 
Detection of the rcscIL-12 protein
 41
Detection of the rcscIL-12 in the established cell line
 24
 24
 25
 26
 
 
 
 23
 
 
 
 
PBMC isolation
Detection of the rcscIL-12 bioactivity
Western blot
 
 
 
 
PBMC pre-stimulation
 
 
 
 
 
 
 
 
 
 
 
 
 
Construction of the rcscIL-12
RESULTS  
 
 
  
Cytotoxicity assay (Rose Bengal Assay)
 
BrdU ELISA
 
 
 
 
 
 
IFN-γELISA
 
 
 
3.3.2 
3.3.3 
3.3.4 
3.3.6 
3.3.5 
3.4 
3.4.1 
 
 
 28
 27
 27
 27
 
 
 
 
 
 
 
 
 
SDS-PAGE   
 
 
 
 
 
 
 
Luciferase assay
 
 
 
Immunofluorescence  
 
 
   Content
 
 
Cloning of the transfected cells
 
nt
onte
9 
8 
  
  
 C
 
B
 
B
I
 
 
 
 
HY
 
 
 
L
IO
GR
AP
 
 
 
 
 
 
 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
59
  
II
 
I
-V
 
 
 
 
 
 
NT
 
HM
E
AC
T
T
A