


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.


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).



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?
1.
2.
3.
4.
5.
6.
7.
8.
Isolate plasmid from bacteria.
Isolate gene of interest from eukaryotic cell.
Treat plasmid with a restriction enzyme.
Treat gene of interest with same restriction enzyme
(this way they have the same sticky ends).
Mix plasmid and gene together. Sticky ends will
hydrogen bond to one another.
DNA Ligase joins the two DNA pieces.
Recombinant plasmid is introduced in bacterial cells.
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.
◦ 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).
◦ 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.



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





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!


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


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.

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
Using the Ti Plasmid to
Make a Transgenic Plant
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.
E The transgenic
plant expresses the
foreign gene. This
tobacco plant is
expressing a firefly
gene.


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
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
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.



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.