University of Pisa
BIOS-Research Doctorate School in BIOmolecular Sciences
Course in Experimental and Molecular Oncology
XXI cycle 2006-2008
RESTORATION OF BRCA1 GENE FUNCTION BY
LENTIVIRUS-VECTOR MEDIATED CELL TRANSDUCTION
Supervisor
Prof.Generoso Bevilacqua
Tutor
Prof. Mauro Pistello
Candidate
There are only two ways to live your life.
One is as though nothing is a miracle.
The other is as though everything is a miracle
-Albert Einstein
INDEX
Abstract... I
Chapter 1: Introduction... 1
1.1 Breast cancer epidemiology... 1
1.2 Risk factors... 3
1.3 Hereditary Breast Cancer... 4
1.4 BRCA1 gene and functional domains... 5
1.5 BRCA1 role in cell homeostasis... 8
1.5.1 DNA repair... 8
1.5.2 Cell cycle checkpoint... 9
1.5.3 Ubiquitination activity... 11
1.6 BRCA1 inactivation Mechanisms and consequences... 12
1.6.1 Breast cancer... 13
1.6.2 Fanconi Anemia... 14
1.7 Hereditary breast cancer prevention... 14
1.8 Gene therapy... 16
1.9 Available tools for gene delivery... 17
1.9.1 Suitable viral vectors... 17
1.10 FIV and FIV vectors... 25
1.11 FIV vectors in viv applications... 27
Chapter 2: Aim of the work... 29
Chapter 3: Materials and methods... 30
3.1 Plasmids used for vector production... 30
3.2 Cloning strategies... 32
3.2.1 Vector optimization... 32
3.2.2 BRCA1 and BRCA1-ires-GFP cloning... 33
3.3 Assessment for vector safety……….... 35
3.5 Vector production and titration... 36
3.6 Transduction protocols... 37
3.7 Analysis of transgene expression... 37
3.8 BRCA1 functional test... 38
Chapter 4: Results... 40
4.1 Vector genome organization... 40
4,2 WPRE increases non-feline cells transduction... 40
4.3 The vector is safe and stable... 43
4.4 LAW34/VSV-G transduces HCC1937 tumor cell line... 45
4.5 Transduction protocol effects on LAW34 efficiency... 47
4.6 BRCA1 and GFP are expressed in transfected cells... 49
4.7 LAW34-BRCA1 and BRCA1-IRES-GFP are efficiently packaged... 52
4.8 BRCA and GFP are expressed in transduced cells... 53
4.9 Transduced BRCA1 restores its activity in HCC1937... 56
4.10 BRCA1-IRES-GFP and LAW34-GFP preferentially transduce primary cells... 59
Chapter 5: Discussion... 62
References... 70
