Post on 02-Jan-2016
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.