Download Lecture 15 Cloning in Mammalian Cells 1. Eukaryotic expression

Lecture 15 Cloning in Mammalian Cells
1. Eukaryotic expression vector
2. Transfection technology
3. Stable transfectants
4. Constitutive and inducible expression
5. Tagging technology
Eukaryotic expression vector
1. It is a shuttle vector, containing ColE1 ori and SV40 origin
2. It has an efficient promoter (e.g. SV40 early promoter, Rous Sarcoma virus promoter, adenovirus major late promoter, or human
cytomegalovirus promoter)
3. It has mRNA processing signals including polyadenylation signal and intervening sequences
Constitutive and inducible expression
1 Constitutive expression (promoters, selection markers, tags)
2. Regulated expression (promoters, inducible systems)
3. Secreted expression (signal sequence)
4. Subcellular localization expression (signal sequence)
5. Generating stable expression cell lines (selection technology)
6. Retroviral expression system Constitutive expression system
pcDNA vector
PCMV: CMV enhancer-promoter
BGHpA: BGH polyadenylation signal and termination sequence
f1 origin SV40 origin SV40 polyadenylation signal
ampicillin resistance gene pUC origin
Invitrogen
Subcellular localization of fusion protein
The pShooter™ vectors are designed to localize recombinant proteins to the nucleus,
mitochondria, endoplasmic reticulum, or cytoplasm.
In addition to the localization signal, each pShooter™ vector contains: Strong
mammalian promoter (CMV or EF-1a) for constitutive expression of your protein of
interest;
C-terminal c-myc epitope for detection with an Anti-myc Antibody;
Bovine Growth Hormone (BGH) polyadenylation site for mRNA stability;
f1 origin for single-stranded rescue of sense DNA;
Neomycin resistance gene for selection of mammalian cells with Geneticin selection
agent;
Ampicillin resistance and pUC origin for selection and maintenance in E. coli;
Each vector is provided with a positive control plasmid. The positive control expresses
GFP and targets it to the same subcellular location as the pShooter™ vector.
pEF-1a uses human elongation factor 1a gene promoter.
Nuclear signal: SV40 large T. Mitochondria signal: cytochrom oxidase VIII signal.
invitrogen
pShooter (ER)
pShooter (mito)
Transfection
Cloned genes can be transfected into cells for
biochemical characterization, mutational
analyses, investigation of the effects of gene
expression on cell growth, investigation of gene
regulatory elements, and to produce a specific
protein for purification.
Transfection: process in which DNA is introduced into eukaryotic cells by chemical or physical methods
Infection: viral-mediated process in which target cells are infected with a virus carrying cloned DNA sequence in its genome
Subcellular localization expression
Nuclear signal: SV40 Large T
Mitochondrial signal: cytochrome oxidase VIII signal
nucleus
mitochondria
Subcellular localization expression
Endoplasmic reticulum signal: SEKDEL
Cytoplasm signal: N/A
ER
cytosol
Transfection technology
Chemical methods
DNA and chemicals form a complex or aggregate which allows cells to take up
DNA via membrane fusion or endocytosis pathway
1. Calcium phosphate
2. DEAE-dextran
3. Cationic liposome
4. Dendrite-based
Physical methods
DNA is introduced directly into the cell
1. Electroporation
2. Biolistic particle delivery
3. Microinjection
Biological methods
Chemical methods
1.  Calcium phosphate: forms an insoluble precipitate with DNA,
not toxic, for both transient and stable transfection (Graham
and van der Eb, 1973)
2.  DEAE-dextran: forms aggregate with DNA, toxic, for transient
transfection, effect promoted by osmotic shock (Vaheri and
Pagano, 1965)
3.  Cationic liposome: trapped DNA as a complex and fused with
the cell, cell toxicity varies, for both transient and stable
transfection (Felgner et al., 1987) 4.  New polymers or chemicals
Lipofection technology
DOTMA (Lipofectin): N-[1-2,3-dioleoyl)propyl]-N,N,N-trimethylammonium chloride
DDAB (LipofectACE): dimethyl dioctadecylammonium bromide
Amidine (CLONfectin): N-t-butyl-N’-tetradecyl-3-tetradecyl-aminopropionamide
DOSPA (LipofectAMINE): 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-
1-propanaminium trifluoroacetate
DOGS (Transfectam): spermine-5-carboxy-glycine dioctadecyl-amide
Lipofection protocol: (i) key reagents. (ii) protocol of transfection.
www.lifetech.com
1.5 mL/$290
Lipids used in lipofection
$
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Commercial kits
Amersham-Pharmacia
Bio-Rad
CLONTECH
5Prime-3Prime
Gene Therapy Systems
Invitrogen Life Technologies Promega
Qiagen
Quantum Biotech Sigma-Aldrich
Stratagene Wako
$
CellPhect Transfection Kit
Cytofectene Reagent CLONfectin
CalciumPhosphate Kit
GenePORTER
Perfect Lipid
Lipofectamine, LipofectACE
Transfectam, Tfx Reagents
Superfect, Effectene
GeneSHUTTLE
Escort, DPOPE
LipoTaxi, Mammalian transfection kit
GeneTransfer HMG-1,2 mixture
Company Brochures
Dendrimers
Dendrimers are highly
branched spherical
molecules in which
branches terminating at
charged amino groups
radiate from a central
core molecule.
Due to controlled
chemical synthesis,
dendrimers have a very
precise size and defined
shape.
Activation of newly synthesized
dendrimers involves removal of
some of the tertiary amines,
resulting in a molecule with a
higher degree of flexibility.
Activated dendrimers yield a
transfection efficiency 2–3
orders of magnitude higher than
non-activated dendrimers. Activated dendrimers assemble
DNA into compact structures
through the interaction of
negatively charged phosphate
groups of nucleic acids with the
positively charged amino groups
of the dendrimers.
PolyFect Transfection
PolyFect Transfection Reagent is a solution of specifically designed
activated-dendrimers. The reagent consists of dendrimer molecules
of a defined spherical architecture with branches radiating from a
central core. The branches terminate at charged amino groups,
which can interact with negatively charged phosphate groups of
nucleic acids. PolyFect Reagent assembles DNA into compact
structures that bind to the cell surface and are taken into the cell by
nonspecific endocytosis. The reagent buffers the pH of the
endosome, leading to pH inhibitition of endosomal nucleases, which
ensures stability of PolyFect–DNA complexes.
PolyFect Technology
Dendrimer Transfection
•  Qiagen (
http://www1.qiagen.com/Products/Transfection/TransfectionReagents/
SuperFectTransfectionReagent.aspx?ShowInfo=1 )
–  Some of the products offered are SuperFect and PolyFect
transfection reagents, which are activated, spherical
dendrimers. They also carry liposome reagents.
•  Dendritic NanoTechnologies, Inc. (http://dnanotech.com/index.php )
–  This company specializes in dendrimers and offers poly
(amidoamine) (PAMAM) dendrimers as transfection reagents,
which they claim to have greater transfection efficiency and less
toxicity than cationic lipid reagents. Marker for assaying the transfection efficiency
Stable transfection using selection markers:
Frequently used selectable markers are
the genes encoding aminoglycoside
phosphotransferase (APH; neoR gene)
or hygromycin B phosphotransferase
(HPH). Other selectable markers include
the genes encoding adenosine
deaminase (ADA), dihydrofolate
reductase (DHFR), thymidine kinase (TK)
or xanthine-guanine phosphoribosyl
transferase (XGPRT; gpt gene).
In some cases, stable transfection
causes a morphological change that can
be used as a selectable trait.
Cell specificity
It is not clear why there is cell type selectivity with regard to a particular transfection reagent.
Physical methods
1. Electroporation: Brief exposure of cells to high-voltage electric pulses forming nanometer-sized pores in the membrane,
very efficient, for transient and stable transfection, no cell
toxicity, applicable to many cell types
(Neumann et al., 1982; Zimmermann et al., 1982)
2. Biolistics: microprojectile bombardment method, gene gun method, particle acceleration method; using gold or tungsten particles to bind DNA, inject directly into cells, tissues, or organelles. Used when all other methods failed
(Sanford et al., 1993)
Physical Transfection - Biolistics
http://www.instr.science.ru.nl/particlegun.html
Physical Transfection - Biolistics
Physical Transfection - Electroporation
http://www.cshl.edu/labs/cline/sce.html
Physical Transfection Technology
• 
http://www.cshl.edu/labs/cline/sce.html
–  This is an article on single cell electroporation. It includes some
interesting pictures of transfection experiments with cells in a tadpole
brain. It mentions that single cell electroporation can not only be used
to insert DNA into a cell, but can also be used for RNA, proteins, dyes,
and drugs. The second part of the article discusses bulk tissue
electroporation and the parameters and conditions that the authors
found to be optimal.
• 
http://dragon.zoo.utoronto.ca/~jlm-gmf/T0701A/biolistic.html
–  This website is a brief overview of biolistic bombardment, which
includes how the method works, how to determine which cells have
taken up the genes by using markers or reporter genes, and the
advantages and disadvantages of biolistics. There is a picture of an
instrument and a closer view of the process.
• 
http://www.instr.science.ru.nl/particlegun.html
–  This website contains information about a biolistic particle gun, with a
picture of the entire instrument, a description of how it is used for
bombardment and applications such as injecting dye-coated particles to
view connections in the brain, and a more detailed picture of how it
works with the procedure of how to use it.
Biological Transfection
•  http://www-ermm.cbcu.cam.ac.uk/04008191h.htm
–  This website contains a very good diagram of how a retrovirus works. It
shows a retroviral vector for MDR-1 (multiple drug resistance 1) binding
to a cell surface, and the RNA genome is reverse transcribed to form DNA
which is transcribed and translated to form the desired protein.
•  http://www.biocompare.com/techart.asp?id=180
–  This website describes retroviral vectors for gene delivery that are
produced by Stratagene. It includes a diagram of production of the vector
that contains the virus and how the cell is infected by retroviral gene
transfer. There is a map of the vector that is being sold, along with other
information about the vector, emphasizing its high efficiency.
Biological Transfection - Retrovirus
http://www-ermm.cbcu.cam.ac.uk/04008191h.htm