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What is transfection?
Broadly speaking, transfection is the process of introducing nucleic acid (DNA or RNA)
into eukaryotic cells by non-viral methods. This introduction of foreign nucleic acids
using various chemical, biological, or physical methods can lead to changes in cell
properties, thereby allowing the study of gene function and protein expression in the
context of cells.
Transfection overcomes the inherent challenges of introducing negatively charged
molecules (for example, the phosphate backbone of DNA and RNA) into cells with
negatively charged membranes. In transfection, the introduced nucleic acid may
temporarily exist in the cell so that it can only be expressed for a limited time without
replication, or it can be stablilized and integrated into the genome of the recipient and
replicated in the host genome.
Transfection Terminology
Transfection Transformation Transduction
Definition
Transfection
commonly refers
to the
introduction of
nucleic acids
into eukaryotic
cells, or more
specifically,
into animal
cells. However,
its present
meaning includes
any artificial
introduction of
foreign nucleic
acid into a cell.
Transformation is
often used to
describe the
non-viral DNA
transfer in
bacteria,
non-animal
eukaryotic cells,
and plant cells.
Transduction is
used to describe
virus-mediated
DNA transfer.
Advantages and Disadvantages of the Different Transfection
Methods
Methods Advantages
Disadvanta
ges
Recommend
ed cells
BOC Sciences
Solutions
Chemical
Lipid
mediated
 High
efficiency-able to
effectively deliver
nucleic acids into
cells and obtain
measurable
changes
 Low
cytotoxicity
 Easy to
use-minimum
steps required;
adapt to high
throughput
systems
 Economi
cal-more active
lipids can reduce
costs and obtain
effective results
 Not
suitable for all
cell
types-some
cell lines
cannot be
transfected by
lipofection
Immortal
cells,
adherent
(attached
) or
suspended
cells
Most of our
products are
based on
cationic
lipid
Calcium
phosphate
 High-effi
ciency
 Cheap
 Can be
applied to
multiple cell
types
 Small
pH changes
(±0.1) may
affect the
efficiency
 The
size and
quality of the
sediment are
critical to
success
Adherent
and
nonadhere
nt cell
lines.
Calcium
Phosphate
Transfection
Reagent
Cationic
Polymers
(e.g., PEI)
 No viral
vector
 Chemi
cal toxicity to
some cell
types
Tumor
cell
lines
PEI
Transfection
Reagent
DEAE-Dextran
 Cheap
 Easy to
perform and
quick
 High
concentrations
of
DEAE-dextran
 Suitable
to a wide range
of cell types
may be toxic
to cells
 Differ
ent
transfection
efficiency in
different cell
types
 Can
only be used
for transient
transfection
 Poor
performance
in primary
cells (usually <
10%)
Magnet
mediated
 Fast
 Improve
transfection
efficiency
through targeted
transport,
especially for
small amounts of
nucleic acids
 Gentle
treatment of cells
 Relativ
ely new
method
 Need
adherent cells;
suspension
cells need to
be fixed or
centrifuged
Immortal
cells,
Adherent
mammalian
cell
lines and
primary
cells
Biological
Viral
mediated
 Very high
gene delivery
efficiency,
95-100%
 Easy to
use
 Effective
for dissociated
cells, slices,
and in vivo
 Most
viruses need
P2
containment
 Risk of
foreign gene
integration
into the host
genome
 Many
viruses are
lytic
Attached
adherent
cells,
stem
cells,
primary
cells
 Virus
DNA Transfection
Reagent
 Lentivira
l Transfection
Reagent
 Polybren
e-Based Virus
Delivery Reagent
 Requir
es packaging
cell line
Immunogenicit
y
 DNA
package size
limit
Physical
Electroporat
ion
 No need
for vector
 Easy and
rapid
 Not alter
target cell
morphology and
functions
 Dema
nds
experimenter
skill, laborious
procedure
 Need
to determine
the optimum
electroporatio
n conditions
 Can
cause cell
death if
transfection is
not performed
under
optimum
conditions
A variety
of cells
(especial
ly cells
that are
difficult
to
transfect
, such as
primary
and stem
cells)
Electroporat
ion Products
Factors affecting transfection efficiency
 Cell passage number
Keep the number of passages<50. In addition, the number of cell passages used in
various experiments should be consistent.
 Cell state
Cells should be grown in a medium suitable for the cell line and supplemented with
serum or growth factors according to survival needs. Contaminated cells and media
must not be used for transfection. If the cells are damaged in any way, they should be
discarded and re-seeded from frozen, uncontaminated stock.
 Cell density
Normally, the confluency of the transfected cells is 40-80%. Too few cells will cause
the culture to grow poorly without cell-to-cell contact. Too many cells can lead to
contact inhibition, which prevents the cells from absorbing foreign DNA.
 Nucleic Acid Quality and Quantity
The plasmid DNA used for transfection should be free of protein, RNA, chemical, and
microbial contamination, and the appropriate final concentration is 0.2-1mg/ml. The
optimal amount of DNA used for transfection depends on the type of DNA, transfection
reagent, target cell line, and the number of cells.
 Cytotoxicity of transfection reagent
When choosing a transfection method or reagent, both the transfection efficiency and
the level of cytotoxicity must be considered. Higher transfection efficiency contributes
to greater success. However, low toxicity methods cannot be ignored, because highly
cytotoxic methods can cause adverse effects in the form of visible morphological
changes and unknown changes in gene expression or stress response pathways.
Transfection classification and application
Transfection is used to study the function and regulation of genes or gene products, to
produce genetically modified organisms, and is used as a powerful analysis tool for
gene therapy methods. According to the time range of the desired experiment and the
goal, transfection can be divided into transient transfection and stable transfection.
 Transient transfection
Transiently transfected cells are usually harvested within 24-96 hours after
transfection. They are usually used to study the short-term expression of genes or
gene products, perform RNA interference (RNAi)-mediated gene silencing, or
rapidly-produce recombinant proteins.
 Stable transfection
In order to obtain long-term gene expression, the DNA vector needs to be integrated
into the host chromosome. This process requires selective screening and clonal
isolation. It is usually used for long-term pharmacological research, gene therapy, or
long-term genetic regulation mechanism research.

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Introduction to Transfection.pdf

  • 1. What is transfection? Broadly speaking, transfection is the process of introducing nucleic acid (DNA or RNA) into eukaryotic cells by non-viral methods. This introduction of foreign nucleic acids using various chemical, biological, or physical methods can lead to changes in cell properties, thereby allowing the study of gene function and protein expression in the context of cells. Transfection overcomes the inherent challenges of introducing negatively charged molecules (for example, the phosphate backbone of DNA and RNA) into cells with negatively charged membranes. In transfection, the introduced nucleic acid may temporarily exist in the cell so that it can only be expressed for a limited time without replication, or it can be stablilized and integrated into the genome of the recipient and replicated in the host genome. Transfection Terminology Transfection Transformation Transduction Definition Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. However, its present meaning includes any artificial introduction of foreign nucleic acid into a cell. Transformation is often used to describe the non-viral DNA transfer in bacteria, non-animal eukaryotic cells, and plant cells. Transduction is used to describe virus-mediated DNA transfer. Advantages and Disadvantages of the Different Transfection Methods Methods Advantages Disadvanta ges Recommend ed cells BOC Sciences Solutions
  • 2. Chemical Lipid mediated  High efficiency-able to effectively deliver nucleic acids into cells and obtain measurable changes  Low cytotoxicity  Easy to use-minimum steps required; adapt to high throughput systems  Economi cal-more active lipids can reduce costs and obtain effective results  Not suitable for all cell types-some cell lines cannot be transfected by lipofection Immortal cells, adherent (attached ) or suspended cells Most of our products are based on cationic lipid Calcium phosphate  High-effi ciency  Cheap  Can be applied to multiple cell types  Small pH changes (±0.1) may affect the efficiency  The size and quality of the sediment are critical to success Adherent and nonadhere nt cell lines. Calcium Phosphate Transfection Reagent Cationic Polymers (e.g., PEI)  No viral vector  Chemi cal toxicity to some cell types Tumor cell lines PEI Transfection Reagent DEAE-Dextran  Cheap  Easy to perform and quick  High concentrations of DEAE-dextran
  • 3.  Suitable to a wide range of cell types may be toxic to cells  Differ ent transfection efficiency in different cell types  Can only be used for transient transfection  Poor performance in primary cells (usually < 10%) Magnet mediated  Fast  Improve transfection efficiency through targeted transport, especially for small amounts of nucleic acids  Gentle treatment of cells  Relativ ely new method  Need adherent cells; suspension cells need to be fixed or centrifuged Immortal cells, Adherent mammalian cell lines and primary cells Biological Viral mediated  Very high gene delivery efficiency, 95-100%  Easy to use  Effective for dissociated cells, slices, and in vivo  Most viruses need P2 containment  Risk of foreign gene integration into the host genome  Many viruses are lytic Attached adherent cells, stem cells, primary cells  Virus DNA Transfection Reagent  Lentivira l Transfection Reagent  Polybren e-Based Virus Delivery Reagent
  • 4.  Requir es packaging cell line Immunogenicit y  DNA package size limit Physical Electroporat ion  No need for vector  Easy and rapid  Not alter target cell morphology and functions  Dema nds experimenter skill, laborious procedure  Need to determine the optimum electroporatio n conditions  Can cause cell death if transfection is not performed under optimum conditions A variety of cells (especial ly cells that are difficult to transfect , such as primary and stem cells) Electroporat ion Products Factors affecting transfection efficiency  Cell passage number Keep the number of passages<50. In addition, the number of cell passages used in various experiments should be consistent.  Cell state Cells should be grown in a medium suitable for the cell line and supplemented with serum or growth factors according to survival needs. Contaminated cells and media must not be used for transfection. If the cells are damaged in any way, they should be discarded and re-seeded from frozen, uncontaminated stock.  Cell density
  • 5. Normally, the confluency of the transfected cells is 40-80%. Too few cells will cause the culture to grow poorly without cell-to-cell contact. Too many cells can lead to contact inhibition, which prevents the cells from absorbing foreign DNA.  Nucleic Acid Quality and Quantity The plasmid DNA used for transfection should be free of protein, RNA, chemical, and microbial contamination, and the appropriate final concentration is 0.2-1mg/ml. The optimal amount of DNA used for transfection depends on the type of DNA, transfection reagent, target cell line, and the number of cells.  Cytotoxicity of transfection reagent When choosing a transfection method or reagent, both the transfection efficiency and the level of cytotoxicity must be considered. Higher transfection efficiency contributes to greater success. However, low toxicity methods cannot be ignored, because highly cytotoxic methods can cause adverse effects in the form of visible morphological changes and unknown changes in gene expression or stress response pathways. Transfection classification and application Transfection is used to study the function and regulation of genes or gene products, to produce genetically modified organisms, and is used as a powerful analysis tool for gene therapy methods. According to the time range of the desired experiment and the goal, transfection can be divided into transient transfection and stable transfection.  Transient transfection Transiently transfected cells are usually harvested within 24-96 hours after transfection. They are usually used to study the short-term expression of genes or gene products, perform RNA interference (RNAi)-mediated gene silencing, or rapidly-produce recombinant proteins.  Stable transfection In order to obtain long-term gene expression, the DNA vector needs to be integrated into the host chromosome. This process requires selective screening and clonal isolation. It is usually used for long-term pharmacological research, gene therapy, or long-term genetic regulation mechanism research.