MOB-20306 Gene technology
Lecture 1 Gene transfer to Mammalian cells and gene therapy
Steps for gene transfer to mammalian cells:
1. Clone gene
2. Manipulate gene in vitro
3. Return gene into organism or single cells (presented most obstacles)
Why gene transfer to mammalian cells?
Research / study gene function:
- Introduce mutated copy of gene to replace normal wild type (functional) gene (‘knockouts’).
- Add extra copies of wild type gene to over-express gene product.
- Tracking experiments using reporter genes to gain information about the localization and
interaction of the protein of interest.
Biotechnology / Use cells as bioreactor:
- Raise transgenic animals that will produce protein of interest (pharmaceuticals).
Gene therapy:
- The insertion of a gene into an individual's cells and tissues to treat a hereditable disease
(whereby a deleterious mutant allele is replaced) with a functional gene.
Complement: proof that a certain gene causes a disease.
Introducing genes in cells is easier, lets focus on that first.
Starting a cell line:
Isolate individual cells by disrupting the extracellular matrix and cell junctions
- Proteolytic enzymes trypsin & collagenase to digest matrix
- EDTA solution to chelate Ca2+ on which cell-cell adhesion depends
- Disrupt cellular matrix by digesting the protein connections by using proteolytic enzymes and
take away Ca2+.
Mammalian cells require a solid surface that is coated with material
they can adhere to (e.g. poly-lysine or extracellular matrix
components)
- This property can be used to obtain specific cell types based on
surface properties and their binding to antibodies (e.g. FACS)
Isolate a certain cell type, 2 ways:
1. FACS = fluorescence-activated cell sorter.
- Antibody coupled to a fluorescent dye to label specific cells
- Droplets containing single cells are given a negative or positive
charge, depending on whether the cell is fluorescent
- And deflected by an electric field into collection tubes according to
their charge.
2. Micro-dissection from tissue slices
- Thin section of tissue under microscope, recognize specific cells,
laser beam cuts around region of interest, second laser beam used to catapult selected
region into container.
Cell lines:
- Cell lines can be most easily generated from cancer cells.
- Cell cultures directly prepared from tissues of an organism are: primary cell cultures
- Repeated culturing can result in a cell line (immortalized by mutations).
- Growth factors are needed to stimulate replication of specific cell types.
- Cells in culture remain differentiated.
, - Cells have to be maintained in cultures
- Tumor cells can only be maintained, normal cells stop growing after a while
- Growth factors (hormones) have to be added for growth.
Many cell lines were derived from tumors. All of them are capable of indefinite replication in culture
and express at least some of the special characteristics of their cell’s origin.
Different methods to get a DNA construct in a cell. Which method is used also depends on the cell
type and application.
1. Ca2+-phosphate co-precipitation
2. Electroporation
3. Lipofection
4. Viral vectors
- Adenovirus (ds DNA virus)
- Adeno-associated virus (ss DNA virus)
- Retrovirus (ss RNA virus)
1st DNA mutations were on viruses.
60s Discovery that in the course of transforming a cell from the normal to the neoplastic phenotype,
the SV40 integrated their genetic information covalently, stably and heritably in to the genomes of
the target cells putative vectors for gene transfer
70s Knowledge on virus biology allowing the modification of its genome to replace and incorporate
coding sequences and effective expression in infected cell. In genetic engineering reverse
transcriptase is used to make an artificial gene of cDNA s (no introns) (requirement of reverse
transcriptase packaged in retrovirus particle to initiate copying into DNA)
1973 Efficient Ca mediated DNA transfer to cells a way
to transfect cells with recombinant DNA vector to
produce viral vectors in vivo using helper cell lines
1. Gene transfer by Ca2+-phosphate co-
precipitation
DNA forms a precipitate around divalent cations, this
precipitate is effectively taken up by cells. Derived from
bacteria transformation.
Isolate DNA from adenovirus particles. Put the DNA in
solution and add CaCl2. Calcium + phosphate =
precipitation. add to cells cells make the virus.
they could show that all the genetic information to make a particle is on the
DNA.
2. Gene transfer by electroporation
Grow the culture without salt in the medium, add DNA, give them an
electroshock of 2500 volt, that shock creates pores in the membrane, allows
DNA to go in. bacteria are ready to die, but because you give them a rich
medium they can recover. then selection based on a marker that you have
introduced.
Stable expression means integration in the genome so that the DNA persists in
all cells derived from the initial few cells that were transformed.
Mammalian cell transformation : integration in genome, so it has not only to
pass the plasma membrane but also the nuclear envelope. Bacterial
transformation : use of plasmid.
If you want to integrate DNA into an eukaryote = linearize plasmid. That
integrates more easier.
, 3. Gene transfer by lipofection
You have to cross plasma membrane that consists of
phospholipids = DNA needs to be packed in a vesicle of lipids.
Vesicle can merge/fuse with membrane.
Mix DNA and lipids = lipid bilayer around DNA = add to cells =
DNA also needs to pass the nucleus envelope and integrate in
the genome.
This can happen very easily if the DNA is linear.
4. Viral vectors
Viruses carry little more than nucleic acid. Viruses are very
diverse, but what they have in common is that they can transfer
their genome in the host cells.
Viral life cycle
virus enters the cell, releases its DNA, DNA can be
transcribed (can lead to production of proteins or DNA can be
replicated = more viruses), DNA can also go into the genome.
- Hypothetical virus:
dsDNA
Single capsid protein
- To reproduce:
Enter host , Replicate, Transcribe and translate (Coat
protein), Assemble, Escape from host
Viruses infect cells efficiently by membrane fusion, pore
formation or membrane disruption.
Lytic viruses kill the host after they copied their genome.
The DNA of the virus integrates in the genome of the host or
exists as episome.
A non-enveloped virus usually leaves an infected cell by lysing it,
an eveloped virus can leave the cell by budding. When capsid is
packaged with viral genome, it is called a nucleocapsid.
Enveloped virus is surrounded by lipid bilayer with virus
encoded transmembrane proteins.
Summary: Viral vectors
- infect cells efficiently:
by membrane fusion, pore formation, or endosomal membrane
disruption
- lytic vs non-lytic
- maintained in cell nucleus:
integrate in the host genome or as episome.
From virus to viral vector
- How much DNA can be inserted in vector
- If we use this, does it integrate in the genome or not
- Does it trigger immunity = you don’t want the body to react with an
immune reaction.
, Life cycle of a retrovirus :
Retrovirus has an RNA genome packed in an capsid.
Needs the enzyme reverse transcriptase. This
enzyme can make a complementary copy of
DNA of the RNA strand. RNA has to be
reversed transcribed to become DNA. Only
this organism has this enzyme. The double
stranded DNA can integrate in the genome of
the host cell and transcribed = copies
generated.
If you want to integrate your gene of interest in the genome, you have to take other things out.
Retro viral vector
LTRs are needed for copying the genome and are promotors. Gag, pol, env, are the genes coding for
necessary things the virus needs. These genes are removed and cloned in separate plasmids.
Replace the genes by multiple cloning sites often contain restriction sites, they are unique for
cloning, multiple cloning sites allow to clone the gene of interest in vector.
Plasmids can only be used in E.coli.
Packaging cell line is needed to get the gene of interest back into a virus particle. Packaging cell lines
are mammalian cells that have taken up the plasmids that contain the necessary genes for viruses,
and start to express the genes coding for those proteins. express gag, pol and envelope protein.
They want to build viruses but have nothing to pack. They will produce viruses that will contain only
your gene of interest and not the gag pol and envelope gene.
Limitations
- Your gene cannot be larger than the space that
is allowed by removing gag pol and env.
Lecture 1 Gene transfer to Mammalian cells and gene therapy
Steps for gene transfer to mammalian cells:
1. Clone gene
2. Manipulate gene in vitro
3. Return gene into organism or single cells (presented most obstacles)
Why gene transfer to mammalian cells?
Research / study gene function:
- Introduce mutated copy of gene to replace normal wild type (functional) gene (‘knockouts’).
- Add extra copies of wild type gene to over-express gene product.
- Tracking experiments using reporter genes to gain information about the localization and
interaction of the protein of interest.
Biotechnology / Use cells as bioreactor:
- Raise transgenic animals that will produce protein of interest (pharmaceuticals).
Gene therapy:
- The insertion of a gene into an individual's cells and tissues to treat a hereditable disease
(whereby a deleterious mutant allele is replaced) with a functional gene.
Complement: proof that a certain gene causes a disease.
Introducing genes in cells is easier, lets focus on that first.
Starting a cell line:
Isolate individual cells by disrupting the extracellular matrix and cell junctions
- Proteolytic enzymes trypsin & collagenase to digest matrix
- EDTA solution to chelate Ca2+ on which cell-cell adhesion depends
- Disrupt cellular matrix by digesting the protein connections by using proteolytic enzymes and
take away Ca2+.
Mammalian cells require a solid surface that is coated with material
they can adhere to (e.g. poly-lysine or extracellular matrix
components)
- This property can be used to obtain specific cell types based on
surface properties and their binding to antibodies (e.g. FACS)
Isolate a certain cell type, 2 ways:
1. FACS = fluorescence-activated cell sorter.
- Antibody coupled to a fluorescent dye to label specific cells
- Droplets containing single cells are given a negative or positive
charge, depending on whether the cell is fluorescent
- And deflected by an electric field into collection tubes according to
their charge.
2. Micro-dissection from tissue slices
- Thin section of tissue under microscope, recognize specific cells,
laser beam cuts around region of interest, second laser beam used to catapult selected
region into container.
Cell lines:
- Cell lines can be most easily generated from cancer cells.
- Cell cultures directly prepared from tissues of an organism are: primary cell cultures
- Repeated culturing can result in a cell line (immortalized by mutations).
- Growth factors are needed to stimulate replication of specific cell types.
- Cells in culture remain differentiated.
, - Cells have to be maintained in cultures
- Tumor cells can only be maintained, normal cells stop growing after a while
- Growth factors (hormones) have to be added for growth.
Many cell lines were derived from tumors. All of them are capable of indefinite replication in culture
and express at least some of the special characteristics of their cell’s origin.
Different methods to get a DNA construct in a cell. Which method is used also depends on the cell
type and application.
1. Ca2+-phosphate co-precipitation
2. Electroporation
3. Lipofection
4. Viral vectors
- Adenovirus (ds DNA virus)
- Adeno-associated virus (ss DNA virus)
- Retrovirus (ss RNA virus)
1st DNA mutations were on viruses.
60s Discovery that in the course of transforming a cell from the normal to the neoplastic phenotype,
the SV40 integrated their genetic information covalently, stably and heritably in to the genomes of
the target cells putative vectors for gene transfer
70s Knowledge on virus biology allowing the modification of its genome to replace and incorporate
coding sequences and effective expression in infected cell. In genetic engineering reverse
transcriptase is used to make an artificial gene of cDNA s (no introns) (requirement of reverse
transcriptase packaged in retrovirus particle to initiate copying into DNA)
1973 Efficient Ca mediated DNA transfer to cells a way
to transfect cells with recombinant DNA vector to
produce viral vectors in vivo using helper cell lines
1. Gene transfer by Ca2+-phosphate co-
precipitation
DNA forms a precipitate around divalent cations, this
precipitate is effectively taken up by cells. Derived from
bacteria transformation.
Isolate DNA from adenovirus particles. Put the DNA in
solution and add CaCl2. Calcium + phosphate =
precipitation. add to cells cells make the virus.
they could show that all the genetic information to make a particle is on the
DNA.
2. Gene transfer by electroporation
Grow the culture without salt in the medium, add DNA, give them an
electroshock of 2500 volt, that shock creates pores in the membrane, allows
DNA to go in. bacteria are ready to die, but because you give them a rich
medium they can recover. then selection based on a marker that you have
introduced.
Stable expression means integration in the genome so that the DNA persists in
all cells derived from the initial few cells that were transformed.
Mammalian cell transformation : integration in genome, so it has not only to
pass the plasma membrane but also the nuclear envelope. Bacterial
transformation : use of plasmid.
If you want to integrate DNA into an eukaryote = linearize plasmid. That
integrates more easier.
, 3. Gene transfer by lipofection
You have to cross plasma membrane that consists of
phospholipids = DNA needs to be packed in a vesicle of lipids.
Vesicle can merge/fuse with membrane.
Mix DNA and lipids = lipid bilayer around DNA = add to cells =
DNA also needs to pass the nucleus envelope and integrate in
the genome.
This can happen very easily if the DNA is linear.
4. Viral vectors
Viruses carry little more than nucleic acid. Viruses are very
diverse, but what they have in common is that they can transfer
their genome in the host cells.
Viral life cycle
virus enters the cell, releases its DNA, DNA can be
transcribed (can lead to production of proteins or DNA can be
replicated = more viruses), DNA can also go into the genome.
- Hypothetical virus:
dsDNA
Single capsid protein
- To reproduce:
Enter host , Replicate, Transcribe and translate (Coat
protein), Assemble, Escape from host
Viruses infect cells efficiently by membrane fusion, pore
formation or membrane disruption.
Lytic viruses kill the host after they copied their genome.
The DNA of the virus integrates in the genome of the host or
exists as episome.
A non-enveloped virus usually leaves an infected cell by lysing it,
an eveloped virus can leave the cell by budding. When capsid is
packaged with viral genome, it is called a nucleocapsid.
Enveloped virus is surrounded by lipid bilayer with virus
encoded transmembrane proteins.
Summary: Viral vectors
- infect cells efficiently:
by membrane fusion, pore formation, or endosomal membrane
disruption
- lytic vs non-lytic
- maintained in cell nucleus:
integrate in the host genome or as episome.
From virus to viral vector
- How much DNA can be inserted in vector
- If we use this, does it integrate in the genome or not
- Does it trigger immunity = you don’t want the body to react with an
immune reaction.
, Life cycle of a retrovirus :
Retrovirus has an RNA genome packed in an capsid.
Needs the enzyme reverse transcriptase. This
enzyme can make a complementary copy of
DNA of the RNA strand. RNA has to be
reversed transcribed to become DNA. Only
this organism has this enzyme. The double
stranded DNA can integrate in the genome of
the host cell and transcribed = copies
generated.
If you want to integrate your gene of interest in the genome, you have to take other things out.
Retro viral vector
LTRs are needed for copying the genome and are promotors. Gag, pol, env, are the genes coding for
necessary things the virus needs. These genes are removed and cloned in separate plasmids.
Replace the genes by multiple cloning sites often contain restriction sites, they are unique for
cloning, multiple cloning sites allow to clone the gene of interest in vector.
Plasmids can only be used in E.coli.
Packaging cell line is needed to get the gene of interest back into a virus particle. Packaging cell lines
are mammalian cells that have taken up the plasmids that contain the necessary genes for viruses,
and start to express the genes coding for those proteins. express gag, pol and envelope protein.
They want to build viruses but have nothing to pack. They will produce viruses that will contain only
your gene of interest and not the gag pol and envelope gene.
Limitations
- Your gene cannot be larger than the space that
is allowed by removing gag pol and env.
