Download Transfection by Ca-phosphate Co

Transfection of
Mammalian Cells
MBIOS 520/420
October 6, 2005
Gene Manipulation
• There are three steps to study the function of gene:
I. Identify and clone the gene.
II. Alter the gene to study its function.
(create a knockout or specific mutation)
III. Re-Introduce the altered gene back into the
organism and analyze the phenotype.
• We’ve already covered ways to achieve Step I
• How do we accomplish Step II & III?
Altering a Gene
• There are several ways we can alter a gene
• We can insert a transposon or marker gene to knock it out
• Or we can make specific point mutations that will change the
codons (and thus the amino acids) of the gene/protein:
• This is called site-directed mutagenesis (your book calls this
oligonucleotide directed mutagenesis
Site-Directed Mutagenesis
Let’s say we want to change
a single codon in exon 2 of
Gene X from AAT to ACC.
We have Gene X in a
plasmid, so we create a
primer identical to part of
exon 2, but with the CC pair
we want instead of AT.
We anneal this to Gene X
and do PCR around the
plasmid (similar to inverse
PCR).
The copies that are made
have incorporated the
mutations in the primer,
changing the codon to
ACC.
ACC
Primer w/
changed codon
AAT
Plasmid
w/ Gene X
Denature & anneal
mutant primer
PCR w/
mutant
primer
Product
after 1st
cycle
Mutant
product
after many
cycles
Mammalian Transfection Techniques
I. DEAE Dextran
II. Calcium-Phosphate Co-Precipitation
III. Electroporation
IV. Microinjection
V. Liposome-Mediated Uptake
VI. Viral Vectors
Mammalian Transfection Techniques
I. DEAE Dextran
II. Calcium-Phosphate Co-Precipitation
III. Electroporation
IV. Microinjection
V. Liposome-Mediated Uptake
VI. Viral Vectors
Transfection
• Once we’ve created a mutant, we need to complete Step III
and introduce it into a cell
• Mammalian cells have defenses against accepting foreign
DNA, designed to protect against viruses
• Early methods involved coupling DNA to positively charged
DEAE-dextran, which “stuck” to cells and entered via
endocytosis
• Most of the DNA was destroyed by the cells’ defenses, so the
method was inefficient
• However, when DNA is precipitated with calcium phosphate,
cells take it in much more efficiently
Transfection by Ca-phosphate Co-precipitation
• In early experiments,
researchers precipitated viral
DNA & added it to
mammalian cell culture
• 100X more viral particles
were produced than when
DEAE-dextran was used
• Because these early
experiments used viruses,
the process was called
transfection:
(transformation-infection)
• Efficiency is still 1/1000 cells
Transfection
• Just like in yeast, we need selectable markers to detect which
cells took up DNA
• For mammals, one of the earliest selectable markers used
was thymidine kinase (tk), since tk- cells had been isolated
• tk allows cells to “salvage” free pyrimidines and convert them
into thymine or adenine nucleotides
• a different enzyme, HPRT, can salvage purines
• aminopterin is a drug that blocks nucleotide synthesis; thus
cells with aminopterin must rely on tk & HPRT activity
• Basis of selection is co-transfection: the phenomenon that
cells transfected with two DNA fragments by Ca precipitation
will usually take up both
Co-Transfection & tk selection
In this experiment, tk gene
and a globin gene are
precipitated via Caphosphate & introduced into
mammalian tk- cells.
Cells are grown on HAT
medium to select for tk+.
HAT MEDIUM
H = hypoxanthine (allows
HPRT to produce purines)
A = aminopterin (blocks
nucleotide synthesis)
T = thymidine (allows tk to
produce pyrimidines)
When cells are isolated &
Southern blots performed,
cells have integrated both
globin & tk genes into their
genomes.
NOTE: We could
do the same
thing for HPRTcells.
High Protein Expression Through Gene Amplification
• Very few cells will actually integrate new DNA into their
genome, but many will transiently express introduced DNA
• neor, a resistance gene to neomycin drug G418 is the most
commonly used selectable marker
• When researchers want to study a protein (to determine its
structure or make antibodies), they need it in large quantities
• DHFR (dihydrofolate reductase) is a critical metabolic enzyme
for creating nucleotides and is inhibited by methotrexate (Mtx)
• Some cells can survive methotrexate treatment by amplifying
the DHFR gene through replication/recombination
• Researchers can attach a gene of interest to DHFR and it will
be amplified along with it, making many copies & lots of protein
Gene Amplification
DHFR
Put both Gene X and DHFR
in vector with a strong
promoter (ex:
cytomegalovirus or CMV)
Transfect DHFR- cells with the
vector & grow on
nucleoside-free medium.
Apply increasing
concentrations of Mtx. Only
a few cells will survive.
Mtx
Those that survive will have
large numbers of vector
DNA amplified
Mtx
Grow these cells & isolate
large amount of Protein X.
Mtx
Mammalian Transfection Techniques
I. DEAE Dextran
II. Calcium-Phosphate Co-Precipitation
III. Electroporation
IV. Microinjection
V. Liposome-Mediated Uptake
VI. Viral Vectors
Electroporation
• Calcium phosphate coprecipitation does not work in
every cell type (ex:
lymphocytes)
• Electroporation uses an
electrical pulse that punches
holes in the plasma
membranes of cells so DNA
can enter
• This method is very efficient,
but usually kills > 50% of the
cells because it is damaging
Mammalian Transfection Techniques
I. DEAE Dextran
II. Calcium-Phosphate Co-Precipitation
III. Electroporation
IV. Microinjection
V. Liposome-Mediated Uptake
VI. Viral Vectors
Microinjection of DNA
• Uses a computer-controlled needle to inject DNA directly into
the nucleus of a cell
• Very reliable, but only be performed on one cell at a time
Mammalian Transfection Techniques
I. DEAE Dextran
II. Calcium-Phosphate Co-Precipitation
III. Electroporation
IV. Microinjection
V. Liposome-Mediated Uptake
VI. Viral Vectors
Liposome-Mediated Gene Transfer
• Artificial lipid vesicles
(liposomes) can be created by
forming a bilayer around DNA
• These capsules adhere to the
cell membrane and fuse into it
• Making liposomes is
complicated, but available
commercially
Mammalian Transfection Techniques
I. DEAE Dextran
II. Calcium-Phosphate Co-Precipitation
III. Electroporation
IV. Microinjection
V. Liposome-Mediated Uptake
VI. Viral Vectors
Viral Vectors for Gene Transfer
• Viruses have the natural ability to successfully introduce DNA
into foreign cells
• Normal plasmid vectors are modified by adding the viral
genome, with gene of interest replacing the viral late genes
• Without the late genes, these viruses cannot replicate, so a
helper virus (lacking early genes) is co-transformed with the
plasmid
• Transformed cells produce both viruses carrying gene of
interest
• Virus is isolated & transformed into new cells, which are unable
to produce viruses (late genes missing) but produce the gene
of interest
SV40 Viral Vector Use
In this experiment, tk gene
and a globin gene are
precipitated via Caphosphate & introduced into
mammalian tk- cells.
Bacculavirus Vector Use
In this experiment, tk gene
and a globin gene are
precipitated via Caphosphate & introduced into
mammalian tk- cells.
Mammalian Gene Knockouts
In this experiment, tk gene
and a globin gene are
precipitated via Caphosphate & introduced into
mammalian tk- cells.
Using PCR to Detect Gene Knockouts
In this experiment, tk
gene and a globin
gene are precipitated
via Ca-phosphate &
introduced into
mammalian tk- cells.