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

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.

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2008
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

   

 

 

 

 

 

 

 

 

 

 

  

  

  

  

  

  

 

        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