Samenvatting MOB-21306
[LECTURE 1] Gene transfer to mammalian cells & Gene therapy
For gene transfer to mammalian cells, there are three important steps:
1. Cloning of the gene of interest
2. Manipulating the gene in vitro (usually in eppendorf)
3. Returning the gene into an organisms or single cells
The first two steps are fairly common and standard now, but the third step is still quite
difficult.
Why do we want to transfer genes
to mammalian cells? Well, there are
three main reasons:
1. Research & studying the
function of a gene: e.g. if
you are interested in finding
the underlying genes for a
particular genetic disease
2. Biotechnology, using the
cells as a bioreactor
3. Gene therapy (always
human), biotechnology is animals or plants
Putting genes back into single cells. First you need a cell line, because you can’t just put the
gene back in a random cell. That means that you need to start a cell line by isolating cells from
a tissue. The first step is disrupting the extracellular matrix, which can be done with:
- Proteolytic enzymes like trypsin & collagenase, to digest the matrix
- EDTA solution, to bind Ca2+. Ca2+ is necessary for cell-cell adhesion
*mammalian cells require a solid surface that is coated
with material that they can adhere to, because they
don’t like to swim in a medium. This can be something
like polylysine, or substances that would normally be in
extracellular matrix. Some cells have compounds that
would make them adhere to such surfaces. These cell-
specific bindingsites can be used to specifically isolate
these cells or bind them to antibiotics.
One way of labelling cells to isolate them is the FACS
(Fluorescence-activated cell sorter):
You can obtain cells from random tissues and then
isolate specific cells from those cell-mixes.
,A less complicated way is to use lasers
and beams to cut specific cells from a
tissue that you want to investigate.
This is based on something that we can
see under a microscope.
So now you have your cells, time to make a
cell line:
Ideally, these cell lines are derived from
cancer cells, because they divide
continuously. So it is more easy to create a
cell line.
The cell lines directly prepared from tissues
are called primary cell cultures, as they will
only divide a certain number of times, after
which they die (unless mutations occur that make them an immortalized cell line). We see
often that we need growth factors to maintain the survival of the cultured cells. When specific
cell lines can be isolated or grown specifically and the cells will remain in a differentiated
status (so based on how far they were developed / differentiated in the original tissue).
Transforming a cell line with gene of interest
Several methods are available to get the gene of interest into the cell:
1. Ca2+ phosphate co-precipitation
2. Electroporation, so giving the cells an electric shock which makes them take up the
plasmids
3. Lipofection, with membrane vesicles that contain DNA and fuse these with the cells
4. Viral vectors, so using virusses that are modified in such a way that they are no longer
harmful but can be used to introduce genes (this is what virusses do, they introduce
genes. This is how they replicate.)
Using reverse transcriptase, a copy of RNA can be made into DNA. BUT it can also be used to
take the introns out of a sequence, so that only the exons / coding sequence remains and put
this into a cDNA (copy DNA).
Human genes have very long introns, which makes it complicated to transform these genes
into other organisms. But using reverse transcriptase, the introns can be removed so only the
coding regions are left – resulting in a smaller part of genetic material for transformation.
,Classical gene transfer via Ca2+ as follows:
DNA was isolated from Adenovirus
particles, introduced into eppendorf with a
buffer and CaCL2, this creates particles.
DNA sticks to the particles, which are plated
on a cell culture and washed. After few days
the medium (NOT THE CELLS) are then
sampled and inside this medium were now
virus particles. Which means that you can
introduce DNA and obtain virus particles.
(just the virus DNA could get into the cells,
replicate and become new virusses).
Gene transform by electroporation:
A plasmid with the gene of interest is prepared,
prefarably with a selectable marker gene that
can later be used to identify which cells have
taken up the plasmid. The plasmid is then mixed
with cultured cells and electrocuted, so that the
cells can take up the plasmids through holes in
the cellmembrane. DNA from the plasmid can
then enter the nucleus and some of it will be
integrated in genome. Cells that express the DNA
and the selectable marker can be selected for =
stable expression.
HOWEVER, transient expression is also possible, so some cells might have
taken up the plasmid including gene of interest & marker, but not
integrated in the genome.
With lipofection there are lipid molecules and DNA with gene of interest.
These lipids will form vesicles that can contain the DNA, such vesicles can
easily adhere and enter into cells, after which some of the DNA might be
integrated into the genome.
, Viral Vectors are extremely
efficient in transforming cells.
That’s what they do.
There are many different types
of virusses, including RNA and
DNA virussues. Some are double
stranded DNA, others are single
strand DNA. Not all of them are
useful for viral vectors.
IMPORTANT: Viral vector is
different from a virus, as a viral
vector CAN NOT replicate itself
into a virus / pathogenic version,
so it is harmless.
Simple life cycle of a virus that uses double
stranded DNA:
It has a single coat protein on the outside, so very
simple. It enters cell, releases DNA and that
needs to replicate, for multiplying the virus. But
the DNA also needs to be transcribed, so from
the DNA, RNA is made, which is translated to
make virus particles that are necessary to
become new virusses that can leave the cell and
infect new cells. (but this is simplified)
The way in which virusses infect cells also differs
between virus species.
The HIV and influenza are envelope virusses that
have the virus particles with the genetic material
surrounded by a membrane.
Virusses that don’t have an envelope cannot
fuse with the membrane and need to be taken
up through endocytosis, forming a ‘vesicle’ after
which the virus uses the vesicle to move
through the cell and escaping it when it is ready
to release the DNA or RNA.
[LECTURE 1] Gene transfer to mammalian cells & Gene therapy
For gene transfer to mammalian cells, there are three important steps:
1. Cloning of the gene of interest
2. Manipulating the gene in vitro (usually in eppendorf)
3. Returning the gene into an organisms or single cells
The first two steps are fairly common and standard now, but the third step is still quite
difficult.
Why do we want to transfer genes
to mammalian cells? Well, there are
three main reasons:
1. Research & studying the
function of a gene: e.g. if
you are interested in finding
the underlying genes for a
particular genetic disease
2. Biotechnology, using the
cells as a bioreactor
3. Gene therapy (always
human), biotechnology is animals or plants
Putting genes back into single cells. First you need a cell line, because you can’t just put the
gene back in a random cell. That means that you need to start a cell line by isolating cells from
a tissue. The first step is disrupting the extracellular matrix, which can be done with:
- Proteolytic enzymes like trypsin & collagenase, to digest the matrix
- EDTA solution, to bind Ca2+. Ca2+ is necessary for cell-cell adhesion
*mammalian cells require a solid surface that is coated
with material that they can adhere to, because they
don’t like to swim in a medium. This can be something
like polylysine, or substances that would normally be in
extracellular matrix. Some cells have compounds that
would make them adhere to such surfaces. These cell-
specific bindingsites can be used to specifically isolate
these cells or bind them to antibiotics.
One way of labelling cells to isolate them is the FACS
(Fluorescence-activated cell sorter):
You can obtain cells from random tissues and then
isolate specific cells from those cell-mixes.
,A less complicated way is to use lasers
and beams to cut specific cells from a
tissue that you want to investigate.
This is based on something that we can
see under a microscope.
So now you have your cells, time to make a
cell line:
Ideally, these cell lines are derived from
cancer cells, because they divide
continuously. So it is more easy to create a
cell line.
The cell lines directly prepared from tissues
are called primary cell cultures, as they will
only divide a certain number of times, after
which they die (unless mutations occur that make them an immortalized cell line). We see
often that we need growth factors to maintain the survival of the cultured cells. When specific
cell lines can be isolated or grown specifically and the cells will remain in a differentiated
status (so based on how far they were developed / differentiated in the original tissue).
Transforming a cell line with gene of interest
Several methods are available to get the gene of interest into the cell:
1. Ca2+ phosphate co-precipitation
2. Electroporation, so giving the cells an electric shock which makes them take up the
plasmids
3. Lipofection, with membrane vesicles that contain DNA and fuse these with the cells
4. Viral vectors, so using virusses that are modified in such a way that they are no longer
harmful but can be used to introduce genes (this is what virusses do, they introduce
genes. This is how they replicate.)
Using reverse transcriptase, a copy of RNA can be made into DNA. BUT it can also be used to
take the introns out of a sequence, so that only the exons / coding sequence remains and put
this into a cDNA (copy DNA).
Human genes have very long introns, which makes it complicated to transform these genes
into other organisms. But using reverse transcriptase, the introns can be removed so only the
coding regions are left – resulting in a smaller part of genetic material for transformation.
,Classical gene transfer via Ca2+ as follows:
DNA was isolated from Adenovirus
particles, introduced into eppendorf with a
buffer and CaCL2, this creates particles.
DNA sticks to the particles, which are plated
on a cell culture and washed. After few days
the medium (NOT THE CELLS) are then
sampled and inside this medium were now
virus particles. Which means that you can
introduce DNA and obtain virus particles.
(just the virus DNA could get into the cells,
replicate and become new virusses).
Gene transform by electroporation:
A plasmid with the gene of interest is prepared,
prefarably with a selectable marker gene that
can later be used to identify which cells have
taken up the plasmid. The plasmid is then mixed
with cultured cells and electrocuted, so that the
cells can take up the plasmids through holes in
the cellmembrane. DNA from the plasmid can
then enter the nucleus and some of it will be
integrated in genome. Cells that express the DNA
and the selectable marker can be selected for =
stable expression.
HOWEVER, transient expression is also possible, so some cells might have
taken up the plasmid including gene of interest & marker, but not
integrated in the genome.
With lipofection there are lipid molecules and DNA with gene of interest.
These lipids will form vesicles that can contain the DNA, such vesicles can
easily adhere and enter into cells, after which some of the DNA might be
integrated into the genome.
, Viral Vectors are extremely
efficient in transforming cells.
That’s what they do.
There are many different types
of virusses, including RNA and
DNA virussues. Some are double
stranded DNA, others are single
strand DNA. Not all of them are
useful for viral vectors.
IMPORTANT: Viral vector is
different from a virus, as a viral
vector CAN NOT replicate itself
into a virus / pathogenic version,
so it is harmless.
Simple life cycle of a virus that uses double
stranded DNA:
It has a single coat protein on the outside, so very
simple. It enters cell, releases DNA and that
needs to replicate, for multiplying the virus. But
the DNA also needs to be transcribed, so from
the DNA, RNA is made, which is translated to
make virus particles that are necessary to
become new virusses that can leave the cell and
infect new cells. (but this is simplified)
The way in which virusses infect cells also differs
between virus species.
The HIV and influenza are envelope virusses that
have the virus particles with the genetic material
surrounded by a membrane.
Virusses that don’t have an envelope cannot
fuse with the membrane and need to be taken
up through endocytosis, forming a ‘vesicle’ after
which the virus uses the vesicle to move
through the cell and escaping it when it is ready
to release the DNA or RNA.
