Transformation/Transfection

Transfection-Transfer of non-viral genetic material into eukaryotic cells.

Infection/ Transduction- Transfer of viral genetic material into cells.

Transformation- Transfer of genetic material into bacterial and plant cells.

Important Terms:

Stable transfectants: Cells that have integrated foreign DNA in their genome.

Transient transfectants: Foreign DNA does not integrate in the genome but genes are expressed for a limited time (24–96 hours).

Important Terms:

Recall that bacteria have a circular genome as well as a small circular piece of DNA called a plasmid.

These plasmids can replicate themselves inside of bacterial cells. ◦ They also usually contain genes for antibiotic resistance.

When we want to put a gene into a bacterial cell, we put the gene into the plasmid and then put the plasmid into the bacteria.

But what about more complex organisms?

Bacterial Plasmids

1. Isolate plasmid from bacteria.2. Isolate gene of interest from eukaryotic cell.3. Treat plasmid with a restriction enzyme.4. Treat gene of interest with same restriction enzyme (this

way they have the same sticky ends).5. Mix plasmid and gene together. Sticky ends will

hydrogen bond to one another.6. DNA Ligase joins the two DNA pieces.7. Recombinant plasmid is introduced in bacterial cells.8. As the bacteria replicate, they will replicate the

plasmids. As they go about their normal functions, they will produce the protein of the gene that we have

introduced.

Transformation Steps

◦ Instead of putting the gene into a plasmid and putting the plasmid into a bacterial cell, we can

instead put the gene into a virus and have the virus put the gene into the bacterial cell's circular chromosome.

◦ This is an application that is being researched for its uses in humans to treat specific cells (since certain viruses only infect certain cells).

Transduction

◦ In 2007, researchers in London reported that they successfully (albeit modestly) treated a rare form of blindness caused by a mutation in a single gene (the gene codes for a protein that is important in producing light absorbing pigments in the eye). Most importantly, the patient suffered from no side effects.

◦ More recently, researchers in France used a modified HIV to introduce genes to correct a rare brain disorder (Adrenoleukodystrophy) in two boys.

Transduction in Humans

Yeast Artificial Chromosomes

Purpose:◦ Cloning vehicles that propogate in eukaryotic cell hosts as

eukaryotic Chromosomes◦ Clone very large inserts of DNA: 100 kb - 10 Mb

Features: YAC cloning vehicles are plasmids Final chimeric DNA is a linear DNA molecule with telomeric ends: Artificial Chromosome

Additional features:◦ Often have a selection for an insert◦ YAC cloning vehicles often have a bacterial origin of DNA

replication (ori) and a selection marker for propogation of the YAC through bacteria.

◦ The YAC can use both yeast and bacteria as a host

Yeast Articial Chromosomes

The production of either random or specific mutations in a piece of cloned DNA. Typically, the DNA will then be reintroduced into a cell or an organism to assess the results of the mutagenesis.

Mutant allelle with a vector Recombination – swap position with normal gene

in a genome. This creates a directed mutant We can use this to discover the function of a

normal gene!

In Vitro Mutagensis w/ Yeast

Cassette of Gene information

consist of DNA sequences which code for an enzyme called transposase. Transposase is able to cleave a transposon from the genome, transport it to a new location, and reinsert it into the genome (conservative transposition)

http://www.public.iastate.edu/~jzhang/Transposition.html

Green Flurescent Protein (GFP)- reporter of gene expression

Transposon Technology (Yeast)

Transformation - changing the phenotype of an organism by the addition of foreign DNA to is genome

And transgenic plants are generated by introducting foreign DNA into a plant tissue and regnerating plants contain the foreign DNA.

Transgenic Plants

Agrobacteria: ◦ carry Ti-plasmid◦ induce tumor formation (crown gall tumor)◦ in wound site of plants◦ produce opines (nopaline or octopine)

Ti-plasmid has two parts:◦ T-DNA the part of DNA that is exported into the

plant cell and integrated into the plant genome◦ vir region: encode proteins involved in this

transfer, but stays within the bacteria

Agrobacteria tumefaciens mediated plant transformation

Here's a schematic diagram that is detailed, although the exact mechanism of transfer is highlighted by the question mark:

E The transgenic plant expresses the foreign gene. This tobacco plant is expressing a firefly gene.

A A Ti plasmid is inserted into an Agrobacterium tumefaciens bacterium. The plasmid carries a foreign gene.

B The bacterium infects a plant cell and transfers the Ti plasmid into it. The plasmid DNA becomes integrated into one of the cell’s chromosomes.

C The plant cell divides, and its descendants form an embryo.

D The embryo develops into a transgenic plant.

Using the Ti Plasmid to Make a Transgenic Plant

A solution containing a cloned gene is injected into the nucleus of a cell.

This technique is especially useful for inserting genes into newly fertilized eggs, since the haploid nuclei of the sperm and egg are relatively large

Animals - Microinjection

Figure: Insertion of new DNA into embryonic cells. Here, DNA (from cloned genes) is injected into the pronucleus of a mouse egg.

DNA fragments may be incorporated directly into cells by incubating the cells in a solution designed to make them “drink” the new DNA in.

The chances of a DNA fragment being incorporated into the chromosomes in this way are relatively small.

DNA is usually mixed with another gene, such as a gene encoding resistance to a particular antibiotic, that enables the rare cells that do incorporate the DNA to “identify themselves” by surviving under culture conditions that kill all the other cells

Animals - Transfection

Genetically Modified Animals• The pig on the left is transgenic for a yellow fluorescent protein; its

nontransgenic littermate is on the right

A high-voltage pulse “pushes” the DNA into the cells.

Animals - Elecroporation

Diagram of the method of gene therapy using electroporation

Inactivate specific genes within an organism and determine the effect this has on the functioning of the organism.

Individual genes are responsible for making a particular protein; thus inactivation of a single gene causes the deletion of that protein.

Genes are knocked out by changing the region of the gene that codes for the protein; this is done by cloning the gene, manipulating it in bacteria, and injecting it into the nuclei of embryonic stem cells which are then placed in the developing test organisms.

Knockout Technology