Lecture 1: Gene transfer to mammalian
cells
Gene transfer to Mammalian cells
1. Isolate & clone gene of interest (cloned in plasmids and multiplied/manipulated ALWAYS in
E. coli)
2. Manipulate sequence of gene in vitro
3. Return the altered gene back into organism or single cells
Gene transfer has become a routine tool for studying gene structure and function: it is used to
identify the regulatory sequences that control gene expression
Why gene transfer to mammalian cells?
• Research – Study gene function: through knockout, over-expression or reporter gene
(localization), provide evidence
• Biotechnology – use cells as bioreactor: transgenic animals will produce protein of interest
• Gene therapy: 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
Return altered gene into Single cells
Starting a cell line
Primary cell cultures: Cell cultures directly prepared from tissues of an organism
Secondary cell cultures: cell cultures that are sub-cultured from a primary culture
Cell strain: a lineage of cells originating from one initial primary culture with a limited lifetime
Cell line: immortal cells as they underwent some genetic changes allowing them to grow unlimited
Starting and maintaining a cell line:
• Isolate individual cells by disrupting the extracellular matrix and cell-junctions: dissociate
cells from tissues
o Proteolytic enzymes trypsin & collagenase to digest/destroy matrix
o EDTA solution to chelate Ca2+ on which cell-cell adhesion depends
• Mammalian cells require a solid surface that is coated with material they can adhere to in
the Petri dish
o e.g., polylysine or extracellular matrix components
o This property can be used to obtain specific cell types based on surface properties
and their binding to antibodies (e.g., FACS – antibodies for specific cells)
, ▪ Some cells bind specifically to specific coatings → sort → isolate specific
cells for a specific cell line
• Cell lines can most easily be generated from tumors/cancer cells.
o They are capable of indefinite replication (unlimited cell divisions) in culture and
express at least some of the special characteristics of their cells of origin.
• Growth factors are needed to stimulate replication of specific cell types
o Cells in culture remain differentiated
• Repeated cell culturing can result in a cell line → immortalized by mutations
Techniques to sort cells
FACS: Fluorescence-activated cell sorter → Cells are sorted based on their fluorescence
• Antibody bound to cells & labeled with fluorescent dye to label specific cells/epitopes
• Droplets containing single cells are given a negative or positive charge, depending on
whether the cell is fluorescent
• Delivers purified cells
o And deflected by an electric field into collection tubes according to their charge
• Start a very specific cell culture
Micro-dissection from tissue slices
• Cells are recognized morphologically (shape/size) that you want to isolate
• Laser beam cuts around region of interest cells and can be dropped into an Eppendorf to be
cultured
• Gravity or second laser to obtain cells
Methods for introducing DNA constructs into mammalian cells: transformation
methods
1. Ca2+ phosphate co-precipitation: Get DNA into cells (mammalian/bacteria)
a. Cells efficiently take up DNA when it is in the form of a precipitate with calcium
phosphate → divalent cations (Ca2+/Mg2+) promote DNA uptake into bacteria
b. DNA contains lot of phosphate → calcium + DNA → DNA precipitate → add DNA
precipitate to cells → DNA is taken up by the cell
2. Electroporation: Apply voltage
a. Mix cells and DNA → Electric shocks on cells → holes in membrane → Cells take up
DNA into cytoplasm directly through holes in membrane (made through shocks) →
integration in genome of cell
i. Not through vesicles, as passage through the endocytic vesicles might
destroy or damage DNA
, b. Stable expression means integration in the genome so that the DNA persists in all
cells derived from the initial few cells that were transformed (linear DNA) – selection
i. Transient expression: Circular DNA less likely to integrate in the DNA
c. No salts: will kill de cell – apply rich medium: take care that the cells will survive –
apply selection medium: stable expression (linear DNA)
3. Lipofection: DNA is packed in a micelle/liposome. Plasmid-liposome complex-mediated gene
transfer
a. DNA (-) is mixed with a lipid solution (+) → micelles are formed → attach to cells (-)
and fuse into plasma membrane (endocytosis) → DNA inserted in
cytoplasm → integrated in the genome → selection
b. Advantages:
i. Lack proteins → non-immunogenic
ii. Can carry exogenous material of essentially unlimited size
iii. Cannot replicate/recombine to form infection agents
c. Disadvantages:
i. Low transduction efficiency as compared to viral vectors
4. Viral vectors: Infect cells efficiently by membrane fusion, pore formation or
membrane disruption – non-lytic
a. Adenovirus (ds DNA virus)
b. Adeno-associated virus (ss DNA virus)
c. Retrovirus (ss RNA virus)
Figure 1. Gene transfer by Ca2+ phosphate co-precipitation
, Viral vectors
Viral gene transduction: introduction of new genes into mammalian cells by packaging them into
virions (= viral vector) The virus is used to insert the transgene into a cell instead of their own genes.
• Infection: Infect cells efficiently by
o Membrane fusion – pore formation –
(endosomal) membrane disruption
o Natural route of entry
• Transfection: introducing nucleic acids into eukaryotic
cells by nonviral methods
• Non-lytic enveloped viruses
o Non-enveloped viruses usually leave an infected
cell by lysing it, but non-lytic enveloped viruses
do not kill the cell → Cell remains alive
o Viruses with an envelope do not kill the cell
when they leave → keep the cell alive and keep
producing
• Maintained in cell nucleus: integrate in the host genome
or as episome
o Long terminal repeats (LTR) needed for
integration
From virus to viral vector:
- The number of nucleic acids that can be inserted in the virus particle
is fixed
o You cannot make the genome size of the virus larger
o When you want to insert your transgene into a virus
particle, other viral genes have to be taken out
- Those viruses should not trigger the immune system of the
host/patient
o Viruses that can escape from the immune system are
preferred
o Coat proteins of virus trigger the immune system
- Some viruses integrate in the genome whereas other stay out of the
genome (episomes)
Viral vectors:
• DNA Adenovirus: maintained as extrachromosomal dsDNA, non-enveloped
o DNA in nucleus as extracellular molecule (episomes)
o Advantages over retroviral vectors:
▪ Most human cell types are susceptible to adenoviral infection and are
subject to efficient transduction
▪ Infects and replicates in dividing and non-dividing cells
▪ Stable and resistant to physical manipulations
▪ Adenoviral cycle does not require integration into the host genome
▪ Promotor of choice can be used (tissue selective expression)
cells
Gene transfer to Mammalian cells
1. Isolate & clone gene of interest (cloned in plasmids and multiplied/manipulated ALWAYS in
E. coli)
2. Manipulate sequence of gene in vitro
3. Return the altered gene back into organism or single cells
Gene transfer has become a routine tool for studying gene structure and function: it is used to
identify the regulatory sequences that control gene expression
Why gene transfer to mammalian cells?
• Research – Study gene function: through knockout, over-expression or reporter gene
(localization), provide evidence
• Biotechnology – use cells as bioreactor: transgenic animals will produce protein of interest
• Gene therapy: 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
Return altered gene into Single cells
Starting a cell line
Primary cell cultures: Cell cultures directly prepared from tissues of an organism
Secondary cell cultures: cell cultures that are sub-cultured from a primary culture
Cell strain: a lineage of cells originating from one initial primary culture with a limited lifetime
Cell line: immortal cells as they underwent some genetic changes allowing them to grow unlimited
Starting and maintaining a cell line:
• Isolate individual cells by disrupting the extracellular matrix and cell-junctions: dissociate
cells from tissues
o Proteolytic enzymes trypsin & collagenase to digest/destroy matrix
o EDTA solution to chelate Ca2+ on which cell-cell adhesion depends
• Mammalian cells require a solid surface that is coated with material they can adhere to in
the Petri dish
o e.g., polylysine or extracellular matrix components
o This property can be used to obtain specific cell types based on surface properties
and their binding to antibodies (e.g., FACS – antibodies for specific cells)
, ▪ Some cells bind specifically to specific coatings → sort → isolate specific
cells for a specific cell line
• Cell lines can most easily be generated from tumors/cancer cells.
o They are capable of indefinite replication (unlimited cell divisions) in culture and
express at least some of the special characteristics of their cells of origin.
• Growth factors are needed to stimulate replication of specific cell types
o Cells in culture remain differentiated
• Repeated cell culturing can result in a cell line → immortalized by mutations
Techniques to sort cells
FACS: Fluorescence-activated cell sorter → Cells are sorted based on their fluorescence
• Antibody bound to cells & labeled with fluorescent dye to label specific cells/epitopes
• Droplets containing single cells are given a negative or positive charge, depending on
whether the cell is fluorescent
• Delivers purified cells
o And deflected by an electric field into collection tubes according to their charge
• Start a very specific cell culture
Micro-dissection from tissue slices
• Cells are recognized morphologically (shape/size) that you want to isolate
• Laser beam cuts around region of interest cells and can be dropped into an Eppendorf to be
cultured
• Gravity or second laser to obtain cells
Methods for introducing DNA constructs into mammalian cells: transformation
methods
1. Ca2+ phosphate co-precipitation: Get DNA into cells (mammalian/bacteria)
a. Cells efficiently take up DNA when it is in the form of a precipitate with calcium
phosphate → divalent cations (Ca2+/Mg2+) promote DNA uptake into bacteria
b. DNA contains lot of phosphate → calcium + DNA → DNA precipitate → add DNA
precipitate to cells → DNA is taken up by the cell
2. Electroporation: Apply voltage
a. Mix cells and DNA → Electric shocks on cells → holes in membrane → Cells take up
DNA into cytoplasm directly through holes in membrane (made through shocks) →
integration in genome of cell
i. Not through vesicles, as passage through the endocytic vesicles might
destroy or damage DNA
, b. Stable expression means integration in the genome so that the DNA persists in all
cells derived from the initial few cells that were transformed (linear DNA) – selection
i. Transient expression: Circular DNA less likely to integrate in the DNA
c. No salts: will kill de cell – apply rich medium: take care that the cells will survive –
apply selection medium: stable expression (linear DNA)
3. Lipofection: DNA is packed in a micelle/liposome. Plasmid-liposome complex-mediated gene
transfer
a. DNA (-) is mixed with a lipid solution (+) → micelles are formed → attach to cells (-)
and fuse into plasma membrane (endocytosis) → DNA inserted in
cytoplasm → integrated in the genome → selection
b. Advantages:
i. Lack proteins → non-immunogenic
ii. Can carry exogenous material of essentially unlimited size
iii. Cannot replicate/recombine to form infection agents
c. Disadvantages:
i. Low transduction efficiency as compared to viral vectors
4. Viral vectors: Infect cells efficiently by membrane fusion, pore formation or
membrane disruption – non-lytic
a. Adenovirus (ds DNA virus)
b. Adeno-associated virus (ss DNA virus)
c. Retrovirus (ss RNA virus)
Figure 1. Gene transfer by Ca2+ phosphate co-precipitation
, Viral vectors
Viral gene transduction: introduction of new genes into mammalian cells by packaging them into
virions (= viral vector) The virus is used to insert the transgene into a cell instead of their own genes.
• Infection: Infect cells efficiently by
o Membrane fusion – pore formation –
(endosomal) membrane disruption
o Natural route of entry
• Transfection: introducing nucleic acids into eukaryotic
cells by nonviral methods
• Non-lytic enveloped viruses
o Non-enveloped viruses usually leave an infected
cell by lysing it, but non-lytic enveloped viruses
do not kill the cell → Cell remains alive
o Viruses with an envelope do not kill the cell
when they leave → keep the cell alive and keep
producing
• Maintained in cell nucleus: integrate in the host genome
or as episome
o Long terminal repeats (LTR) needed for
integration
From virus to viral vector:
- The number of nucleic acids that can be inserted in the virus particle
is fixed
o You cannot make the genome size of the virus larger
o When you want to insert your transgene into a virus
particle, other viral genes have to be taken out
- Those viruses should not trigger the immune system of the
host/patient
o Viruses that can escape from the immune system are
preferred
o Coat proteins of virus trigger the immune system
- Some viruses integrate in the genome whereas other stay out of the
genome (episomes)
Viral vectors:
• DNA Adenovirus: maintained as extrachromosomal dsDNA, non-enveloped
o DNA in nucleus as extracellular molecule (episomes)
o Advantages over retroviral vectors:
▪ Most human cell types are susceptible to adenoviral infection and are
subject to efficient transduction
▪ Infects and replicates in dividing and non-dividing cells
▪ Stable and resistant to physical manipulations
▪ Adenoviral cycle does not require integration into the host genome
▪ Promotor of choice can be used (tissue selective expression)
