Transfection of Novel DNAs into Mammalian CellsWhy Bother - PowerPoint PPT Presentation

Title: Transfection of Novel DNAs into Mammalian CellsWhy Bother


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Transfection of Novel DNAs into Mammalian
CellsWhy Bother?

• Promoter Analyses
• Reporter Gene Assays
• Protein Function
• Overexpression
• Reduced Activity
• Mis-expression
• Mutation Analyses

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Two Types of Transfections

• Transient
• Short term
• No selection required
• Generally a small percentage of cells have
  transfected DNA

• Stable
• Permanentgenerates a new cell line
• Requires selection
• All cells have transfected DNA

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Production of a stable cell line requires a
Dominant Selectable Marker
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Approaches for Gene Delivery Getting Past the
Cell Membrane

• Biochemical
• Calcium Phosphate Co-precipitation
• Liposomes
• Physical
• Microinjection
• Electroporation
• Biolistic Particle Delivery
• Infections
• Retrovirus
• Adenovirus
• Adeno-associated virus

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Approaches for Gene Delivery

• Biochemical
• Calcium Phosphate Co-precipitation
• Liposomes

Charge is the Key!
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Approaches for Gene Delivery

• Biochemical
• Calcium Phosphate Co-precipitation

Endocytosis
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Approaches for Gene Delivery

• Biochemical
• Liposomes

Membrane Fusion
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Approaches for Gene Delivery

• Physical
• Microinjection
• Electroporation
• Biolistic Particle Delivery

Disruptions of Membranes is Key!
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Approaches for Gene Delivery

• Infections
• Retrovirus
• Adenovirus
• Adeno-associated virus

Advantages Highly Efficient
Disadvantages Requires Packaging Cell Line and
Laborious
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Differences in Viral Delivery of Transgenes
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Advantages/Disadvantages of Viral Vectors

• Retrovirus
• Requires dividing cells (exception Lentivirus)
• Random Insertion
• Limited insert size (8 Kbp)
• Stable expression
• Adenovirus
• Works with dividing and non-dividing cells
• No integration in host genome
• Relatively large insert size (35 Kbp)
• Transient expression
• Adeno-associated Virus
• Infects dividing and non-dividing cells
• Potential for selective insertion (currently
  random)
• Limited insert size (4-5 Kbp)
• Stable expression
• Difficult packaging strategies

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Promoter Analyses Selection of Reporter Genes

• Typically want a heterologous reporter
• Should be easily measurable
• Soluble assaysgrind up the cells
• Luciferase
• ?-galactosidase
• Chloramphenicol Acetyl Transferase
• Secreted reportersevaluate media
• Growth Hormone
• Secreted Alkaline Phosphatase
• Staining Assayslook at the cells directly
• ?-galactosidase
• Green Fluorescent Protein
• Should not be regulated post-translationally

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Promoter AnalysesAn Example

• Question Is the promoter for the Scalyskin
  (ssk) gene regulated by cAMP?
• Tools/Reagents
• Cells that express the Scalyskin gene skin.
• Plasmids (Vectors) containing promoters linked to
  a measurable reporter (luciferase)
• Scalyskin promoter (ssk-luc)
• How much?
• Positive control (CRE3-TK-luc)
• Negative control (?CRE3-TK-luc)
• Promoterless control (luc)
• Transfection efficiency control (CMV-?gal)
• A transfection vehicle lipid mediated

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Minimal Promoters/Response Elements
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Promoter Example Theoretical Results
Vehicle
cAMP
Luciferase Activity/?-gal Activity
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Protein Analyses

• Promoter Selection
• Promoter that works in cells of choice
• Inducible vs. Constitutive
• Approach depends on question being asked
• Overexpression More activity than usual
• Mis-expression Activity at the wrong time or
  place
• Reduced Expression Loss of activity
• Anti-sense
• siRNA
• Dominant Negative Protein
• Expression of a Mutant Altered activity

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Protein AnalysesAn Example

• Question Does overexpression of Scalyskin
  induce proliferation?
• Tools/Reagents
• Cells that should respond to Scalyskin
  overexpression (skin)
• Plasmid capable of overexpressing Scalyskin
• Negative control (empty vector)
• Positive control (vector encoding something known
  to induce proliferation in skin cells)
• Proliferation assay
• Transfection approach (stable vs. transient
  transfection)
• Assay to quantify overexpression

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Assessment of Protein Function--Vectors
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Protein Analyses ExampleTheoretical Results
50
100
0
Proliferating Cells
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An Example of Inducible Expression Tet Off
Premise I cant get stable cell lines that
express protein Deadly. I think that
expression of this protein is lethal.
Constitutive Promoter
No Cells
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An Example of Inducible Expression Tet Off
PMin
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Sample Questions Transfections

• You have a gene that is only expressed in breast
  cancer and you think that it is regulated by
  estrogen. How do you test this?
• You want to determine if Protein X regulates the
  promoter for Gene Y. How do you test this?
• You believe that Gene Z is an oncogene (causes
  transformation of normal cells). How do you test
  this?