Download Molecular biotechnology

Molecular biotechnology
Harald H Sitte, MD
Medical University of Vienna, Austria
Learning outcomes
• Overview of cell biology
• Gene expression
• Recombinant deoxyribonucleic acid (DNA)
technology
• Recombinant DNA (rDNA)
The procaryotic cell
Figure 1. Cross section (aftificial) through a bacterial cell. The surface structures
(fimbriae and flagella) are not essential structures but allow the cells to adhere and to
move
Crommelin Ed. Pharmaceutical Biotechnology. 3rd edition,
Taylor & Francis CRC Press, 2007
The eucaryotic cell
Nuclear membrane
mRNA
DNA
Golgi bodies
Mitochondria
Endoplasmic reticulum
Cellular membrane
Lodish et al. Molecular Cell Biology 2e.
Scientific American Books; 1990
DNA, RNA, genes and chromosomes
http://www.virtualsciencefair.org/2005/anna5m0/public_html/images/chromosome.gif
The cell cycle
Replication
DNA
daughter
strands
DNA
parent
strand
The cell cycle
Transcription
T
A A
G C
A
T T
C
G
Ribosome
attaches to
mRNA
mRNA
DNA
parent
strand
Single stranded mRNA
travels from DNA
to a ribosome
Translation
Messenger RNA
Codon
Recombinant
DNA technology
Source
DNA
Cloning
vector
Vector DNA
Target
DNA
Enzymatic
fragmentation
Enzymatically
linearize
DNA construct
Join target DNA
and cloning vector
Introduce DNA into host cell
Host cell
Isolate cells with cloned gene
Protein encoded
by cloned gene
Produce protein from cloned gene
Expression plasmid:
eg for a eucaryotic cell
CMV-promoter
Poly-adenylation
signal
Multiple
cloning site
Gene for
selection
(bacterial
selection with
kanamycin,
mammalian
cell selection
with geneticin)
SV40promoter
Target gene
?
Poly-adenylation
signal
Phage introduction
Crommelin Eds, 3rd edition,
Taylor & Francis CRC Press, 2007
Bacterial transformation
1. Naked DNA fragments from disintegrated
cells in the area of a potential recipient cell.
This cell must be of correct genus and be in
a state of competence, allowing the entry of
the DNA fragments
2. Entry of naked DNA into competent cell
3. Some DNA fragments replace (recombine with) original
host cell DNA. The resultant recombinant DNA will now
express the foreign genes it has received and pass them
on to all its offspring
DNA that has not
recombined is
broken down by
enzymes
http://www.learner.org/channel/courses/biology/archive/images/1854_d.html
Non-bacterial transformation
• Chemical methods:
•
•
•
•
Ca2+
CaPO4-precipitation method
Lipofection
Dendrimers
DEAE-Dextran
Ca2+
Ca2+
Non-bacterial transformation
• Physical methods:
• Gene-gun
• Electroporation
• Micro-injection
New techniques towards
drug development
Mass production of
pharmaceutical products
Possible cure of genetic disease
• Sickle cell anaemia
• Cystic fibrosis
Conclusion slide
• Overview of cell biology
– Different cells can be used
• Gene expression
– Cell cycle and DNA
• Recombinant DNA technology
– Getting the cells do what you want
• Recombinant deoxyribonucleic acid (rDNA)
– The basis for development of novel drugs