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.

AAT

Plasmidw/ Gene X

ACC

Primer w/ changed codon

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 Ca-

phosphate & introduced into mammalian tk- cells.

Cells are grown on HAT medium to select for tk+.

HAT MEDIUMH = 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 HPRT- cells.

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

Put both Gene X and DHFR in vector with a strong

promoter (ex: cytomegalovirus or CMV)

Mtx

Mtx

Mtx

DHFR

Transfect DHFR- cells with the vector & grow on

nucleoside-free medium.

Apply increasing concentrations of Mtx. Only

a few cells will survive.

Those that survive will have large numbers of vector

DNA amplified

Grow these cells & isolate large amount of Protein X.

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 co- precipitation 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 Ca-

phosphate & introduced into mammalian tk- cells.

Bacculavirus Vector Use

In this experiment, tk gene and a globin gene are precipitated via Ca-

phosphate & introduced into mammalian tk- cells.

Mammalian Gene Knockouts

In this experiment, tk gene and a globin gene are precipitated via Ca-

phosphate & 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.