Download Manipulating genes and cells (Kap. 10)

Manipulating genes and cells
(Kap. 10)
¾ restriction enzymes and agarose gel electrophoresis
¾ DNA sequencing
¾ nucleic acid hybridization techniques
¾ genomic and cDNA libraries
¾ cloning of DNA
¾ PCR and PCR applications
¾ isolating cells and growing them in culture
¾ protein expression in recombinant cell lines
¾ genetically altered animals and plants
Experimenting with DNA
Traditional crossbreeding
Genetic engeneering
•Transfer of many genes
•Transfer of a single or a few gene
•Genes are not known
•Genes are known
•Large changes in protein expression
•Small changes in protein expression
•Within species
•Between species
DNA
The DNA-helix
In 1953, based on X-ray
diffraction images taken by
Rosalind Franklin and the
information that the bases
were paired, James D.
Watson and Francis Crick
suggested the DNA structure
in the journal Nature.
Experimental evidence for
Watson and Crick's model
were published in a series of
five articles in the same
issue of Nature.
Restriction enzymes, molecular
scissors from bacteria
EcoR1 bound to DNA
Restriction endonucleases cleave DNA
at specific nucleotide sequences
The Nobel Prize in Physiology or
Medicine 1978
"for the discovery of restriction enzymes and their application to problems of molecular genetics"
Werner
Arber
Daniel
Nathans
Hamilton
O. Smith
Gel electrophoresis
to separate DNA molecules
Agarose gel electrophoresis
DNA sequencing (1)
Dideoxy method
DNA sequencing (2)
DNA sequencing (3)
DNA sequencing using fluorescent
dies and a laser detector
DNA sequencer (capillary electrophoresis)
Celera Genomics (Craig Venter) and the
Human Genome Sequencing Consortium
(Francis Collins) sequenced the human
genome
Francis Collins
(NIH NHGRI)
Craig Venter, Celera
Genomics
Strategies for sequencing complex
genomes
Clone-by-clone shotgun sequencing
Whole-genome shotgun sequencing
Important genome sequencing papers
Mouse
Nature. 420: 520 -562. (5 December 2002)
Human
Nature. 409: 860-921. (15 February 2001)
Arabidopsis - First Plant Sequenced
Nature 408: 796-815. (14 December 2000)
Fruit Fly
Science. (24 March 2000) 287: 2185-95.
Roundworm – C. elegans - First Mutlicellular Eukaryote Sequenced
Science. (11 December 1998) 282: 2012-8.
Bacteria - E. coli
Science. 277: 1453-1474. (5 September 1997)
Yeast
Science. (25 October 1996) 274: 546, 563-7.
Bacteria - H. influenzae - First Free-living Organism to be Sequenced
Science. (28 July 1995) 269: 496-512.
There are more than 600 genomes
completey sequenced in 2007
See GOLD (genome online databases at http://www.genomesonline.org)
The future of DNA sequencing
•
In May 2007, James D. Watson, the
co-discoverer of the molecular
structure of DNA, became the first
person to receive his own complete
personal genome sequenced.
•
The cost was less than $1 million
(0.03 cents per base).
•
It took less than two months.
•
It is realistic to expect that within the
next ten years, rapid low-cost
sequencing of human genomes will
become a reality.
•
Æ Pharmacogenomics
•
Æ Genotyping
Hybridization of DNA
Southern blotting (DNA)
Northern blotting (mRNA)
Western blotting (protein)
E. Southern, "Detection of specific sequences
among DNA fragments separated by gelelectrophoresis," J Mol Biol, 98:503, 1975.
A Northern blot shows amount and size of
mRNA of 1 gene present in different tissues
DNA microarrays (DNA chips) look at many
genes (whole transcriptomes) at once
glas or silicium chip
each dot represents DNA
of one gene spotted on the
chip and hybridized with
the 2 probes
->
identify up- or downregulated
genes in different samples
Genes (DNA) can be located on
chromosomes by fluorescence in situ
hybridization (FISH)
6 different DNA probes
2 dots on the maternal, 2
on the paternal
chromosome 5
(chromosomes have
already replicated their
DNA)
mRNA can be located on cells by in
situ hybridization
Radioactively labeled probe,
exposure on X-ray film
Non-radioactive labeled probe,
visible precipitating reaction product
Multi-labeling techniques
different coloured
enzymatic reaction
products:
Different fluorochromes:
Ligation of DNA to form
recombinant DNA molecules in vitro
Some bacteria can efficiently take up
DNA from their surrounding
introduction of DNA into bacteria: transformation
introduction of DNA into eukaryotic cells: transfection
A typical plasmid cloning vector
DNA cloning
DNA ligase inserts a
DNA fragment into a
bacterial plasmid
DNA plasmid is
introduced into a
bacterium and
replicates
Construction of a genomic library to
isolate human genes by DNA cloning
Colony hybridization:
identification of bacterial colonies
containing a particular DNA
Synthesis of cDNA (reverse transcriptase)
•
Many essential molecular
biology techniques (working
with restriction enzymes,
ligations) do not work with
single-stranded mRNA
•
mRNA is rather instable and
rapidly degraded by
ubiqitary RNAses
•
Therefore, the sequence
information from a mRNA
molecule is first transcribed
into a more stable and
clonable DNA copy (cDNA)
Genomic versus cDNA clones
PCR (polymerase chain reaction)
amplifies DNA sequences
The Nobel Prize in Chemistry 1993
Michael Smith
Canada,
for his fundamental
contributions to the
establishment of
oligonucleotide-based, sitedirected mutagenesis and
its development for protein
studies
Kary B. Mullis
USA, for his invention of
the polymerase chain
reaction (PCR) method
PCR machine
Obtaining genomic or cDNA clones by PCR
PCR can be used to detect viral infections
in a blood sample
Real-time or quantitative PCR
Often using SYBRE-Green, a dye that emits fluorescent light only when
bound to double-stranded DNA produced in the PCR-reaction tube
Recombinant protein expression in
cell lines
New recombinant plasmids are
spliced together
Large amounts of protein are
produced in cell culture
An expression vector (plasmid DNA)
MCS (multiple cloning site)
Strong eucaryotic
promoter from the
cytomegalo virus
Resistence
gene for E. coli
Resistence gene
for mammalian cell
lines
From gene to protein and from protein to gene
Fluorescence-activated cell sorting
(FACS) to isolate specific types of cells
FACS
Cells in culture
Fibroblast secrete collagen
Muscle cells contract
Nerve cells make synapses
Epithelial cells make sheets
HeLa cells (cervical cancer from
Henrietta Lacks)
Nomarsky
Phase contrast
CHO-K1 (chinese hamster ovary) cell line
A cell culture lab
Working in cell culture
sterile,
sterile,
sterile!
12-wells
6-wells
24-wells
1536-wells
96-wells
Reporter genes can determine the
pattern of a gene´s expression
When is the gene
expressed?
Where is the gene
expressed?
How much?
Green fluorescent protein (GFP)
Bioluminescence reaction in aequorea
victoria
Green fluorescent protein (GFP) can
identify specific cells in a living animal
About 20 neurons in a
Drosophila embryo
express GFP under
the control of a
neuronal fly promoter
GFP expressed in the eyes of Drosophila
A cell-membrane protein fused to GFP
Fluorescent proteins
Spectra of fluorescent proteins
β-galactosidase as a reporter gene
Section of a fly brain
Genetically engineered organisms
Gene addition: e.g. GFP as a marker
in transgenic animals
Creating a ”Knockout
mouse” using
embryonic stem cells
(ES cells) to
selectively delete a
single gene/protein
The phenotype of this KOmouse can give a hint to the
physiological function of this
protein in the wild-type
organism
Antisense RNA can generate
dominant negative mutations
RNA interference (RNAi) knock-down
Transgenic plants can be made with an
Agrobacterium
A tobacco plant expressing GFP
Bio-pharming (transgenic plants)
Inserting genes that instruct a plant to manufacture
pharmaceutical compounds: plants as drug-producing bioreactors.
Golden rice
Rice expressing 3 transgenes
responsible for provitamin-A
(beta-carotene) synthesis
Gene-pharming (transgenic animals)
mostly used to make human proteins that have medicinal
value. The protein is secreted into the animal's milk, eggs
or blood, and then collected and purified.
Genetic engineering and ethical issues
•therapeutical cloning
•PDS (pre-implantation genetic diagnosis and selection)
•germline cloning