Cell transfection

1
Cell transfection

• Lamia Wahba and Tovah Honor
• March 26, 2007

2
Roadmap

• Basic overview
• Transfection techniques
• Selection markers and fusion genes
• Present Day uses and advances

3
Early Years

• Transfection transformation infection
• The infection of cells by the isolated nucleic
  acid from a virus, resulting in the production of
  a complete virus

4

• Competent cell requirements
• uptake of viral nucleic acid
• replication of viral molecules
• assembly of progeny virus release
• Not required
• ability of host to form a colony
• ability to undergo genetic recombination
  with transfecting DNA

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(1978)
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Modern Day

• Transfection is the process whereby the nucleic
  acid sequences are introduced by either
  biochemical or physical processes.
• Biochemical methods DEAE-dextran, calcium
  phosphate, and liposome-mediated transfection
  methods.
• Physical transfection direct micro-injection of
  materials, biolistic particle delivery and
  electroporation.

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Transfection method 1 DEAE-Dextran

• Facilitates DNA binding to cell membranes and
  entry via endocytosis
• DEAE-D associates with DNA?complex binds to cell
  surface

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Transfection method 1 DEAE-Dextran

• Major variants number of cells, concentration of
  DNA and concentration of DEAE-Dextran
• Only method that cannot be used for stable
  transfections

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Transfection method 2 electroporation

• Use of high-voltage electric shocks to introduce
  DNA into cells
• Cell membranes electrical capacitors unable to
  pass current
• Voltage results in temporary breakdown and
  formation of pores

Harvest cells and resuspend in electroporation
buffer
Add DNA to cell suspensionfor stable
transfection DNA should be linearized, for
transient the DNA may be supercoiled
electroporate
Selection process for transfectant
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Transfection method 2 electroporation

• Variants amplitude and length of electric pulse
• Less affected by DNA concentration-linear
  correlation between amount of DNA present and
  amount taken up
• Can be used for stable transfections

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Transfection method 3 liposome-mediated

• Use of cationic lipids
• chemical and physical similarities to biological
  lipids
• spontaneous formation of complexes with DNA,
  called lipoplexes
• High efficiency for in vitro transfections
• Can carry larger DNA than viruses
• Safer than viruses
• Low in vivo efficiency

http//homepage.usask.ca/sdw132/images/lipid_nano
particle.gif
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Selection markers

• 1 in 104 cells stably integrate DNA

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Large-scale transfection of mammalian cells for
the fast production of recombinant protein

• Phuong Lan Pham, Amine Kamen, and Yves Durocher

• Adaptation of current protocols for higher
  protein output in mammalian cells
• use mammalian cells because of post-translational
  modifications
• use proteins for high-throughput screens,
  clinical uses
• Cells grown in liquid culture instead of adherent
  to subrstrate
• Use CaPi or PEI for high efficiency
• Output milligram to gram amounts

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Palmitic Acid-Modified Poly-L-Lysine for
Non-Viral Delivery ofPlasmid DNA to Skin
Fibroblasts

• Meysam Abbasi, Hasan Uludag? , Vanessa Incani,
  Cori Olson, Xiaoyue Lin, Basüak Acüan Clements,
  Dorothy Rutkowski, Aziz Ghahary, and Michael
  Weinfeld

• Most cationic lipid delivery methods have low in
  vivo efficiency, but many polymers (PEI) are
  toxic
• Added a lipid (PA) used in vesicle trafficking to
  a cationic lipid (PLL)
• higher efficiency of DNA delivery than PLL alone
• less toxic than PEI
• Uses cells endogenous transport system to move
  DNA to nucleus

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Cell microarrays and RNA interference chip away
at gene function

• Douglas B Wheeler, Anne E Carpenter  David M
  Sabatini

• Purpose generate genome-scale libraries of RNAi
  reagents to allow for broad screens in cultured
  cells
• Transfected cell microarrays nucleic acids and
  transfection reagents printed on a standard glass
  slide
• reverse trasnfection

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High-throughput loss-of-function screens using
RNAi cell microarrays.
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How do you make transfected cell microarrays?

• Plasmid DNA in an aqueous gelatin solution
• Lipid transfection reagents
• Total expression levels proportional to amount of
  plasmid DNA used
• Technique is compatible with cotransfection

Ziauddin and Sabatini, Nature 411 (2001)
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An example of a high-density, high-content RNAi
cell-microarray screen in cultured D.
melanogaster Kc167 cells.
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Cell microarrays and RNA interference chip away
at gene function

• Douglas B Wheeler, Anne E Carpenter  David M
  Sabatini

• Why high-throughput RNAi screening??

Implementation of RNAi varies depending on the
organism of choice. For D. melanogaster or C.
elegans, long dsRNAs homologous to the target
mRNA can be used to induce RNARNAi in mammals,
however, is complicated by the interferon
response, which is activated by exogenous dsRNAs
longer than 30 nucleotidesDespite rules that
have been established to choose effective shRNAs
and siRNAs, unknown secondary mRNA structure or
factors render many RNAi constructs unable to
initiate RNAi. Currently, validated whole-genome
collections of mammalian RNAi reagents with at
least one effective RNAi construct per gene are
not yet available.
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MATra - Magnet Assisted Transfection combining
nanotechnology and magnetic forces to improve
intracellular delivery of nucleic acids

• J. Bertram

Use of magnetic particles to improve delivery of
biomolecules, including DNA, into cells
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In Conclusion

• Cell transfection is a widely-used technique with
  many applications
• protein production for clinical or research
  applications
• changing the protein expression profile of a cell
  to assay for the effects of a gene
• overexpression of gene
• RNAi - reduction in expression of gene
• addition of genetic markers to a cell line