Microinjection
Membrane Fusion
TRANSLATION
ssRNA
dsDNA
REVERSETRANSCRIPTION
STABLEINTEGRATION
Virus Particle
RELEASEOF DNA
RELEASEOF DNA
Protein
Protein
mRNA
mRNA
mRNATRANSLATION
Endosome
AdenoviralTransduction
LentiviralTransduction
RELEASEOF RNA
Virus Particle
CAR
CationicPolymers
CationicPolymers
+++
PlasmidDNA+
++
+
+++
+CalciumPhospate
CalciumPhospate
Ca2+
Ca2+Ca2+
PlasmidDNA Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Electroporation
+– +
– +–
PlasmidDNA
PlasmidDNA
mRNA
TRANSLATION
Protein
ds-siRNA
ss-siRNA
RISC
TRANSCRIPTION
Fuse-It-siRNA
siRNA
Fuse-It-mRNA
DEGRADATION
PROTEINEXPRESSION
PlasmidDNA
TransfectionReagent
LYSOSOMALDEGRADATION
Lysosome
Endosome
Lipofection
Transfection of Eukaryotic Cells
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Lentiviruses—a subclass of retroviruses—have the ability to permanently integrate into the genome of the host cell.
Transduction of dividing cell lines and primary, non-dividing cells
Permanent integration and possibility of selection—ideal for stable cell lines
Biosafety level S2 required
Lentiviral Transduction
Replication-deficient adenoviruses are a class of double-stranded DNA viruses that are suitable for transient gene delivery.
Transduction of dividing cell lines and primary, non-dividing cells
Transient only: no nucleotide integration into the host genome
Biosafety level S2 required
Adenoviral Transduction
The cell membrane is permeabilized by a short electric pulse, allowing the nucleo-tides to enter.
Transient and stable transfection of any cell type
Easy and reliable, but high cell numbers needed due to high rates of cell death
Special electroporation device required
Electroporation
With this physical method, the nucleotide solution is directly injected into the nucleus using a fine glass capillary.
High efficiency and controlled nucleotide amount for each cell
Only feasible for special applications (e.g., creating transgenic animals)
Expensive, very time-consuming and a lot of practice and skill required
Microinjection
A mixture of nucleotides, calcium, and phos-phate buffer forms a precipitate that is taken up by the cells via endocytosis.
Inexpensive and simple application
Very sensitive concerning cell constitution, pH, and nucleotide quality / amount
Suitable for most cell lines, but toxic for most primary cells
Calcium Phosphate
The nucleotides are bound to a cationic polymer, such as DEAE-dextran, which is taken up by the cells via endocytosis.
Inexpensive and simple application Easily reproducible after protocol optimization
Highly cytotoxic, therefore not suitable for transfection of sensitive cells and generation of stable cell lines
Cationic Polymers
Complexes of cationic liposomes and nu-cleotides fuse with the cell membrane and release the nucleotides into the cytoplasm.
Easy application and high reproducibility after protocol optimization
Efficiency strongly depends upon the cell type
Uptake of nucleotides mainly by endo-cytosis can reduce efficiency
Lipofection
Molecules are incorporated into liposomal carriers, which fuse with the cell membrane and directly release them into the cytoplasm.
High biocompatibility for sensitive and difficult-to-transfect cells
Transduction of dividing cell lines and primary, non-dividing cells
No interfering processes such as endocytosis or lysosomal degradation
Membrane Fusion
Ca2+
Ca2+Ca2+
+++
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Transfection is defined as the process of inserting nucleic acids (e.g., plasmid DNA, mRNA, or siRNA) into the cyto-plasm of eukaryotic cells. In addition, proteins and nanoparticles such as beads or dyes can be transfected.
In the field of cell biology, transfection is an important and widely used tool for deciphering the function of various genes. This method can be used for gene silencing via RNAi, gene editing via CRISPR/Cas9, as well as overexpression studies using cellular integration of plasmid DNA, mRNA, or proteins.
Choose Your ibidi Product for Your Transfection Assay:
Fuse-It-siRNA
A fusion-mediated siRNA transfection reagent for biocompatible and efficient gene silencing
Fuse-It-mRNA
A reagent for fusion-medi-ated mRNA transfection for immediate analysis of living cells
LifeAct Adenoviral Vectors
Ready-to-use adenoviral vectors for F-actin visuali-zation in sensitive cells
LifeAct Lentiviral Vectors
Lentiviral vectors for easy generation of stable LifeAct- expressing cell lines
