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The
real Cancer fighter
Clare
Fasching
I
spent the morning transfecting expression
constructs into three different cell lines. Tomorrow, I will
collect genomic DNA*, extra-chromosomal DNA
and protein from these cells. I also will process
a fraction of cells by fluorescence-activated cell sorting
(FACS) to check for transfection efficiency, since
the constructs express the jelly fish green fluorescent
protein. ( * terms in italics are defined
below )
My experiments this afternoon were to start the first day
of fluorescence in-situ hybridisation (FISH). This
starts by hybridising chromosomes with specific DNA probes.
Tomorrow I will wash any unbound probe off the slides and
use a biotin/avidin fluorescent detection system
to visualise the location of the plasmid DNA that
has integrated into the genome of these cells.
This is what they will look like after tomorrow’s detection
(hopefully)!
I started my career as a clinical cytogeneticist,
which is someone who analyses chromosomes often for pre-natal
diagnosis. I moved into the cancer field in which genomic
instability features prominently. Looking at chromosomes is
a method of analysing cells for genomic instability,
but it also has many other uses as well.
I am following the movement of a plasmid tag placed within
the telomeres of a cell line that has an active telomere
maintenance mechanism involving inter-telomeric recombination.
These methods are a few I used during the course of my PhD.
I just handed in my thesis this March and plan on starting
a post-doctoral fellowship within the field of genomic instability.
My love of science began at an early age and until I started
University, I thought I wanted to be a veterinarian. After
finishing, I became a clinical cytogeneticist then progressed
into a research laboratory that investigated tumour suppressor
genes.
After moving into cancer research I realised that this was
the career I wanted. I get bored very easily and found that
in research every day was different. I might be repeating
similar experiments, but the question is always different.
I can’t image myself in any other profession.
What do all those technical terms mean?
Transfection: a method of introducing
DNA into mammalian cells.
Expression construct: a piece of
DNA containing a specific gene, which when introduced into
mammalian cells is able to make the specific protein.
Genomic DNA: The DNA found in chromosomes
that comprises all the genes in an organism. Sequencing the
genomic DNA from humans was the basis for the Human Genome
Project.
Extra-chromosomal DNA: DNA that
is not found in chromosomes. There are some cancer cells that
have significant amounts of DNA separate from their chromosomes.
The exact function of this extra-chromosomal DNA is currently
unknown.
Protein: Elements that carry out
all the functions within the cell.
Transfection efficiency: The number
of cells into which a piece of DNA has been introduced. The
higher the number of cells containing introduced DNA, the
better the efficiency of the experiment.
Expression: This term is used to
describe the manufacture of proteins by the cell. Thus, cells
express proteins.
Fluorescence in-situ hybridisation:
This is a method of finding a specific sequence of DNA on
a chromosome. The sequence may have been introduced or may
be part of the genomic DNA.
Hybridisation of Chromosomes: The
two strands of DNA on the chromosomes are separated by high
temperatures. A piece of DNA with a biotin tag attached is
laid on top of the chromosomes and stored at 37oC. The tagged
DNA finds its specific sequence on the chromosome and binds
to it. The separation of the DNA strands is called denaturation
and the binding of complimentary strands is called annealing.
The whole process is called hybridisation. This is the first
step in the Fluorescence in-situ hybridisation experiments.
DNA Probes: Pieces of DNA with specific
sequences that have a biotin tag. This tag allows the location
of the specific sequence of DNA to be identified on a chromosome.
Biotin/avidin fluorescent detection system:
Biotin is a B complex vitamin, which can be attached to DNA.
If this DNA is complementary to specific sequences and is
attached to biotin, it becomes a DNA probe. Avidin is a protein
that binds tightly to biotin. Avidin with a dye that gives
off light at a specific wavelength known as a fluorochrome
attached to it will bind to biotin and allow the location
of that biotin to become visible. Biotin that is attached
to the specific DNA probe and is also bound to Avidin that
is attached to a fluorochrome allows us to locate that specific
DNA sequence on the chromosome.
Plasmid DNA: Bacteria contain circular
pieces of DNA that are extra-chromosomal. These pieces of
DNA can be engineered into an expression construct by inserting
a specific gene.
Genomic instability: The term genomic
is a derivative of genome, which is the term for all the genes
and DNA sequences in an organism. Normally, chromosomes are
copied and separated as the cell divides to create an organism
or maintain tissues such as skin. This process requires a
very stable genome, so every cell has identical genes. Very
rarely genes can start to change, which can lead to changes
in other genes. This can continue until enough changes accumulate
and the cell turns into a cancer cell. These changes in genes
make the genome unstable, which is why we used the term genomic
instability. There are several diseases that are characterised
by many changes in genome including Werner’s syndrome and
Bloom’s syndrome.
Telomeres: A specific sequence of
DNA, TTAGGG in humans that is repeated at the end of each
linear chromosome. This specific repeat sequence binds to
specific Telomere binding proteins that together with the
TTAGGG repeats form a structure to protect the genomic DNA
from changes.
Telomere Maintenance Mechanism:
In normal cells the telomeres get shorter as the cell divides.
When the telomeres become very short the cell “knows” that
it no longer is needed, so it stops dividing. Cancer cells
have made changes that cause the telomeres to be lengthened,
so they never get too short. In other word, the telomeres
are maintained. This tricks the cell into living forever.
Inter-telomeric recombination: One
way the cell lengthens its telomeres is to copy the DNA from
another telomere (inter-telomeric means from another telomere).
The term recombination is used rather than replication, since
this mechanism is normally used by the cell for DNA repair
(see Wikipedia).
Tumour suppressor genes: These are
a set of genes that protect normal cells from becoming cancer
cells.
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