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Microinjection Membrane Fusion TRANSLATION ssRNA dsDNA REVERSE TRANSCRIPTION STABLE INTEGRATION Virus Particle RELEASE OF DNA RELEASE OF DNA Protein Protein mRNA mRNA mRNA TRANSLATION Endosome Adenoviral Transduction Lentiviral Transduction RELEASE OF RNA Virus Particle CAR Cationic Polymers Cationic Polymers + + + Plasmid DNA + + + + + + + + Calcium Phospate Calcium Phospate Ca 2+ Ca 2+ Ca 2+ Plasmid DNA Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Electroporation + – + – + – Plasmid DNA Plasmid DNA mRNA TRANSLATION Protein ds-siRNA ss-siRNA RISC TRANSCRIPTION Fuse-It-siRNA siRNA Fuse-It-mRNA DEGRADATION PROTEIN EXPRESSION Plasmid DNA Transfection Reagent LYSOSOMAL DEGRADATION Lysosome Endosome Lipofection Transfection of Eukaryotic Cells www. .com 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 Ca 2+ Ca 2+ Ca 2+ + + + © ibidi GmbH, V 1.0, 2018 / 02 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

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Page 1: Transfection of Eukaryotic Cells - ibidi...Transient and stable transfection of any cell type Easy and reliable, but high cell numbers needed due to high rates of cell death Special

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

www. .com

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+

+++

© ib

idi G

mb

H, V

1.0

, 201

8 / 0

2

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

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TRANSFECTION PROTOCOL Lipofectamine 3000 Transfection Reagent MCF10A · 2020. 8. 12. · TRANSFECTION PROTOCOL Lipofectamine 3000 Transfection Reagent MCF10A Breast cancer cells Complete

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Protein Production in Insect Cells: Flow Electroporation ... · (co)transfection producing high cell viabilities & transfection efficiencies for a variety of insect cell lines. •The

FuGENE HD Transfection Reagent - Promega commercially available transfection reagents. FuGENE® HD Transfection Reagent can be used in transfection protocols in the presence of serum

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ibidi Application Guide Microscopy With the ibidi Chambers · 3 ibidi Polymer Coverslip The ibidi Polymer Coverslip, which is our most recommended surface, is a thin plastic coverslip

Transfection Optimization Using Expression Analysis/media/Files/BLSS Downloads/BRKS...Transfection Optimization Using Expression Analysis Introduction Optimization of cell transfection

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Transfection · 6800 Medical Sciences: Abrahamet al. A C B ID FIG. 1. Effect ofhumanHOgene transfection on endothelial cell factor VIII antigen andAc-LDLuptake in endothelial cells

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in vitro DNA & siRNA transfection reagent · CPT114 vO (November 2018) 2 jetPRIME® DNA & siRNA Transfection Reagent 1 TRANSIENT DNA TRANSFECTION PROTOCOL 1.1 CELL SEEDING For optimal

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