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Transcript

Cell transfection

By: Àngel Térmens Forcada

Start

Index

Introduction

Transfection types

Transfection methods

Deliveries

Reagents

Conclusion

Introduction to
cell transfection

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Transfection is the process of introducing nucleic acids into eukaryotic cells by non viral methods.

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What is transfection?

The inherent challenge for transfection is introducing negatively charged molecules (e.g., phosphate backbones of DNA and RNA) into cells with a negatively charged membrane.

Using various chemical or physical methods, this gene transfer technology enables the study of gene function and protein expression in a cellular environment.


How does transfection work?

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Physics:

  1. Electroporation
  2. Cell microinjection
  3. Biolistic particle delivery


Chemicals:

  1. Cationic lipids
  2. Nonliposomal reagents
  3. Cationic polymers (chemical reagents)

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Transfection applications

mammalian promoter and enhancer sequences studies

trans-acting proteins
studies

mRNA processing

...as one of the earliest study steps...

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Transfection applications

protein-protein interactions

translation
studies

recombination events

...as one of the earliest study steps...

Deliveries

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DNA

RNA

Transcient and stable

Transcient

DNA

  1. Study gene function and regulation
  2. Mutational analysis and biochemical characterization of gene products
  3. Effects of gene expression on the health and life cycle of cells
  4. Large scale production of proteins for purification and downstream applications

mRNA and Non-coding RNA

  1. Protein knockdown studies
  2. Phenotype analysis
  3. Function recovery
  4. Pathway analysis
  5. In vivo knockdown
  6. Drug target discovery

Transfection types

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Transfection types

Stable
transfection

Transient transfection

Cotransfection

Foreign DNA is either integrated into the cellular genome or maintained as an episomal plasmid.

Unlike transient transfection, stable transfection allows the long-term maintenance of the exogenous DNA in the transfected cell and its progeny.

The introduced nucleic acid exists in the cell only for a limited period of time and is not integrated into the genome.

As such, transiently transfected genetic material is not passed from generation to generation during cell division, and it can be lost by environmental factors or diluted out during cell division.

Cotransfection refers to the simultaneous transfection with two separate nucleic acid molecules, such as plasmid DNA and siRNA. Cotransfection is a common procedure for stable transfection. The plasmid DNA may contain a gene that is easily assayed and acts as a marker.

Stable transfection

  1. Transfected DNA integrates into the genome.
  2. Transfected genetic material is carried stably from generation to generation; genetic alteration is permanent.
  3. Requires selective screening for the isolation of stable transfectants.
  4. Only DNA vectors can be used for stable transfection; RNA by itself cannot be stably introduced into cells.
  5. Single or low copy number of stably integrated DNA results in lower level of protein expression.
  6. Requires 2–3 weeks of selection for the isolation of stably transfected colonies.
  7. Suitable for studies using vectors with inducible promoters.

Transcient transfection

  1. Transfected DNA is not integrated into the genome, but remains in the nucleus.
  2. Transfected genetic material is not passed onto the progeny; genetic alteration is not permanent.
  3. Does not require selection.
  4. Both DNA vectors and RNA can be used for transient transfection.
  5. High copy number of transfected genetic material results in high level of protein expression.
  6. Cells are typically harvested within 24–96 hours of transfection.
  7. Generally not suitable for studies using vectors with inducible promoters.

Cotransfection

Transfection methods

04

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Transfection methods

Calcium phosphate transfection

Cationic lipid transfection

RNAi & siRNA Transfection

Reagents to enable the introduction of DNA into eukaryotic cells via calcium phosphate co-precipitation.


Procedure:

  1. Mix DNA
  2. Incubate
  3. Add the DNA-calcium phosphate
  4. Assay the cells

Specially designed cationic lipids facilitate DNA & siRNA delivery into cells.


Procedure:

  1. Dilutions
  2. Combine to form complexes
  3. Add complexes to cells
  4. Assay cells

Specially designed cationic lipids facilitate DNA & siRNA delivery into cells.



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Transfection methods

Electroporation

In vivo transfection

CRISPR transfection

Electroporation is a physical transfection method that uses an electrical pulse to create temporary pores in cell membranes through which substances like nucleic acids can pass into cells.


Procedure:

  1. Prepare cells
  2. Apply electrical pulse
  3. Return cells to growing conditions
  4. Assay cells

Effective & easy-to-use in vivo RNAi delivery reagents used to achieve phenotypic alternations in animals.

The CRISPR-Cas9 system greatly simplifies genome editing and has great promise in broad applications such as stem cell engineering, gene therapy, tissue and animal disease models, and engineering disease-resistant transgenic plants.


Reagents

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Reagents for continuous cell lines

Neon electroporation

Lipofectamine 2000

Lipofectamine 3000

Lipofectamine LTX

Lipofectamine RNAiMAX

Lipofectamine CRISPRMAX

Invivofectamine 3.0


Reagents for primary and stem cells

Neon electroporation

Lipofectamine 3000

Lipofectamine Stem

Lipofectamine LTX

Lipofectamine RNAiMAX

Lipofectamine CRISPRMAX

Lipofectamine MessengerMAX




Conclusion

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Conclusion

The choice between transient or stable transfection will usually depend on your goals, cell type and the facilities available to you. In short, transient transfection exerts a temporary influence on your cells, whereas stable transfection leads to permanent genetic changes that are usually passed on to future cell progeny.

Webgraphy

ThermoFisher scientist

Altogen Biosystems

Promega

Lonza

Thanks!