Biochemistry 611 Nucleic Acids 83007

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Biochemistry 611 Nucleic Acids8-30-07
Recombinant DNA Technology
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Topics To Be Covered

• Protein Techniques
• Western blotting
• Yeast 2 hybrid
• Single Nucleotide Polymorphisms (SNPs)
• Transfection and Expression in Mammalian Cells
• Discuss vectors
• Factors affecting transfection
• Promoters Enhancers
• Transient vs. Stable Expression
• Delivering DNA to cells
• RNAi
• Expression and Purification of Proteins using
  Bacterial Systems
• Advantages and drawbacks
• Plasmid Vector Systems
• Protein Tags
• Columns and Matrices

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Western Blotting
Method that uses antibodies to detect the
expression of protein(s) Involves separating
protein on SDS-PAGE electrophoresis and transfer
of protein electrophoretically to nitrocellulose
or PDV filters which are then probed with
antibodies.
What is the purpose of SDS?
Maintains protein in denatured state and
neutralizes charge and allows separation based
on size alone

• Sensitivity
• Can detect 0.1 ng of a 50 KDa protein
• Approximately 1000 protein molecules/cell
• Load capacity for gel approx. 150mg/well

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Yeast-2-Hybrid
LacZ blue colonies His3 selection on plates
lacking histidine
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What is Recombinant DNA Technology?

• Altering the genetic makeup of an organism by
• Adding new DNA to the organism or
• changing the DNA that is already present in the
  organism

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Polymorphisms
A polymorphism is a genetic variant that appears
in at least 1 of a population

• Why study polymorphisms?
• Disease diagnosis ? Alzheimer's
• Tissue typing ? donor organs
• Population studies ? long term migration, genetic
  variations

What do polymorphisms have to do with DNA
recombination? Polymorphisms simply represent
genetic changes that occur naturally without
scientific engineering
http//home.comcast.net/john.kimball1/BiologyPage
s/P/Polymorphisms.html
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Single Nucleotide Polymorphisms (SNPs) SNiPs

• DNA sequence variations that occur when a
  single nucleotide in the genome sequence is
  altered
• For a variation to be considered a SNP, it must
  occur in at least 1 of the population
• SNPs make up about 90 of all human genetic
  variation
• occur every 100 to 300 bases along the
  3-billion-base human genome
• Two of every three SNPs involve the replacement
  of cytosine (C) with thymine (T)
• SNPs can occur in both coding (gene) and
  noncoding regions of the genome
• Many SNPs have no effect on cell function, but
  others could predispose people to disease or
  influence
• their response to a drug

http//www.ornl.gov/sci/techresources/Human_Genome
/faq/snps.shtml
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Lots of Ways to Look at SNPs
SamplePreparation
Method of Analysis
Gut, IG, Human Mutation 17475.492 (2001)
Lots of People Looking at SNPs

• The SNP consortium
• Pharmaceutical companies
• NIH
• Various academic researchers

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Transfer and Expression of Foreign Genes in
Mammalian Cells
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Considerations regarding delivering DNA into
mammalian cells

• Virus Mediated
• very efficient (approach 100)
• - Size limitation for virus packaging (2.5 kb
  SV40 6 kb retrovirus)
• - can get rearrangement during virus
  propagation
• - cytopathic effect of virus limits protein
  expression time
• Direct DNA Transfer
• - lower efficiency (5-50)
• less labor intensive then virus mediated

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Advantages and Disadvantages to Expressing
Recombinant Proteins in Mammalian
Expression Systems
Advantages
Disadvantages

• Proper protein folding
• Proper posttranslational modifications
• Recombinant proteins almost always
• accumulate in the correct cellular compartment

• Expensive to do
• Technically more challenging
• Takes more time to do
• Difficult to do on large scale

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Common Features of Mammalian Expression Vectors

• Bacterial Origin of Replication
• Kozak sequence mRNA seq that facilitates
  binding to ribosome (RCCATGG)
• Selectable Markers Neomycin or Kanamycin
  resistance
• Constitutively or Induced protein expression
  (promoter driven)
• Produce large amounts of Protein (Enhancer
  driven)

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Tags Commonly used in Mammalian Expression Systems
What are some uses for these tags??

• Uses include
• Purification
• Visualization

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Considerations in using Mammalian Expression
Vectors

• Type of promoter and enhancer sequence (next
  slide)
• strong vs. weak
• inducible vs. constitutive
• Type of expression
• stable vs. transient
• Amount of expression
• Factors Affecting Transgene Expression
• Number of copies of transgene in cell
• Rate of transcription
• mRNA stability
• Integration position

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Common Promotor/Enhancer Elements
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Common Mammalian Viral Expression Systems
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Schematic of Recombinant Adenovirus
system (Stratagene AdEasy-1 System)
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Other Viral Vector Systems
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Methods of Direct Transfer of DNA into
Mammalian Cells
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• CaPO4 Precipitation
• Compound that precipitates DNA onto cells thus
  allowing the cells to take up DNA by endocytosis
  or phagocytosis
• Factors influencing efficiency
• pH
• Quality of DNA
• Growth State of Cells
• Liposomes

• Nucleic acid is encapsulated in artificial
• lipid membrane vesicles (liposomes) that
• Integrate in phospholipid bilayer
• Advantages
• Relatively high efficiency
• Useable on various cell types
• Can deliver DNA, RNA or Protein
• Disadvantages
• Expensive and can be cytotoxic
• Each batch needs to be optimized

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• Electroporation
• Electroporation capitalizes on the relatively
  weak nature of the cell membranes phospholipid
  bilayer's hydrophobic/hydrophilic interactions
  and its ability to spontaneously reassemble after
  disturbance
• It is thought that a quick voltage shock may
  disrupt areas of the membrane temporarily,
  allowing polar molecules to pass, but then the
  membrane may reseal quickly and leave the cell
  intact.
• Advantages Disadvantages
• ? Can be used on all cell types ? Potential
  cell damage
• ? Up to 80 efficient ? Nonspecific
  transport
• ? Can be done with little DNA (smaller scale)
  ? Needs to be optimized for cell types
• ? Can be performed on intact tissue ? Cost
  of machine
• Microinjection
• Used primarily for inserting DNA into oocytes
• Requires a lot of skill

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Transient Expression
Stable Expression

• Stable gene expression obtained by using
• vectors that confer selection or provides a gene
• essential for survival
• Integrate into the genome - passed onto daughter
  cells
• Exist as episomes (ex. Virus)
• Episomal vectors offer the following benefits
• Accept larger pieces of DNA
• DNA expression is independent of genomic
  integration
• Multiple copies enhance expression

• Short term (12-72hrs) high expression of
  transgene
• High expression scenario is not well tolerated
  by the
• cell and usually results in inhibition of cell
  growth or
• result in the destruction of the vector
• Used for metabolic studies using a reporter gene

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RNAi
Biological mechanism by which double-stranded RNA
(dsRNA) induces gene silencing by targeting
complementary mRNA for degradation
http//www.nature.com/nrg/journal/v2/n2/animation/
nrg0201_110a_swf_MEDIA1.html
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IN NON-MAMMALIAN CELLS Long double-stranded RNAs
(dsRNAs typically gt200 nt) are processed into
20-25 nucleotide (nt) small interfering RNAs
(siRNAs) by an RNase III-like enzyme called Dicer
(initiation step) Then, the siRNAs assemble
into endoribonuclease-containing complexes known
as RNA-induced silencing complexes (RISCs),
unwinding in the process The siRNA strands
subsequently guide the RISCs to complementary RNA
molecules, where they cleave and destroy the
cognate RNA (effecter step) Cleavage of cognate
RNA takes place near the middle of the region
bound by the siRNA strand IN MAMMALIAN
CELLS Introduction of long dsRNA (gt30 nt)
initiates a potent antiviral response that
nonspecifically suppresses gene expression The
mammalian antiviral response can be bypassed by
transfecting small dsRNA (21-23nt) siRNAs or
expressing small hairpin RNAs (shRNAs)
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• In vitro preparation of siRNA
• Chemical synthesis
• In vitro transcription
• Digestion of long dsRNA in vitro by RNase III or
  Dicer
• Introduction of DNA-based vectors and cassettes
  that express siRNAs within cells
• Expression from a plasmid
• Expression from a viral vector
• Expression from a PCR product

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Uses for RNAi

• Targeting viral pathogens (AIDS)
• Targeting disease or symptom-causing genes
  (alpha-Synuclein - Parkinsons disease)
• Removing undesirable gene products
• Selectively killing cancer cells

Paddison PJ, Hannon GJ. Cancer Cell. 2002
Jul2(1)17-23.
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Expression and Purification of Proteins using
Bacterial Systems
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Bacterial Protein Expression
Probably the most used system for expressing and
producing recombinant proteins
Advantages
Drawbacks

• Relatively Simple
• Cheap to Grow
• Flexibility
• Sophistication

• Lack Post-Translational Modifications
• Expressed proteins can be in inclusion
• bodies (insoluble protein aggregates)

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Common Features of Bacterial Expression Plasmids

• Bacterial Origin of Replication
• Promotor/Operator
• Ribosome binding site (RBS)
• Multiple cloning site
• Transcription Terminator
• Selectable Marker

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To get Idea of Versatility Novagen pET vectors
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Some Common Bacterial Expression Protein Tags
There are also tags such as FLAG that can be
purified with antibody columns
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Bacterial Fusion Tags Generally Have
a Protease Cleavage Site to Allow for Tag Removal
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Other Methods for Converting Nucleic Acids into
Proteins

• In vitro translation systems
• Rabbit reticulocyte (RR)
• RR lysates are created by first making rabbits
  anemic (folic acid and phenylhydrazine
  injections). Reticulocytes, lysed osmotically,
  are treated with micrococcal nuclease to destroy
  endogenous RNA
• better for large proteins
• contain exogenous methionine
• sensitive to salts, hemin, Ca2, alcohols and
  detergents
• Wheat germ extract
• Prepared by grinding wheat germ in an extraction
  buffer followed by centrifugation to remove cell
• debris. The supernatant is subjected to
  chromatography that separates endogenous amino
  acids and
• plant pigments from the extract. The extract is
  also treated with micrococcal nuclease to destroy
• endogenous mRNA
• better for smaller proteins
• Needs energy generating system as well as
  spermidine or spermine

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Schematic of In-vitro Translation
ambion web page
RNA generated in vitro utilizes T7, SP6 or T3 RNA
polymerase in a separate uncoupled reaction
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Coupled In-vitro TranscriptionTranslation RR
System
System supplemented with RNA polymerase
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Xenopus Oocytes

• Not as commonly used
• Offers high capacity for translocation
• Extracts give consistent processing of mRNA
• Stability of membranes allow means of isolating
  and verifying translocation products

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Baculovirus Systems

• Utilizes Autographica californica nuclear
  polyhedrosis virus (AcNPV)
• Takes advantage of the viral machinery by
  hijacking the promoters for 2 nonessential but
  highly expressed genes (polyhedron protein and
  p10 gene product) to expression a transgene
• Considerations
• Initial work is time consuming
• Considered very safe (only infects 2 types of
  moths)
• Extremely high yields - up to 30 of protein
  mass may
• be recombinant protein
• Grown at room temp
• Posttranslational modifications and folding
  usually
• successful for eukaryotic proteins

Homologous Recombination in insect cells (sf9)
Screen For plaques
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Proteomics
Proteome (PROTEin complement to a genOME)
Proteomics is a direct analysis of the protein
content, modifications and interactions
Includes such tools as Numerous chromatography
techniques Mass Spectrophotometry Protein
Microarrays
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Proteomics a Quick Overview
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THEORETICAL ANALYSIS

• You are studying a gene that may be linked to a
  disease state
• Where can you go from there???
• Database searches (genomic and proteomic)
• Is the gene highly expressed?
• How big is the mRNA?
• Does the gene product (protein) interact with
  DNA?
• What DNA sequence does the protein bind?
• Does the protein interact with other proteins?
• Where does the protein function?
• Does expression of the protein affect cellular
  viability or function?

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END