Transcript Transformations of Cells

Transformations of
Cells (and Transfections too)
Bacteria, Fungi, Plants, and Animal
Cells & Recombinant DNA
Technology
Transformation
 Transformation (def): the genetic alteration of
a cell resulting from the introduction, uptake
and expression of foreign genetic material
(DNA) in molecular biology
 This can be done to Bacteria, Fungi, Plants,
and Animal cells
Transformation - History
 1928 - Frederick Griffith transforms nonpathogenic
pneumococcus bacteria into a virulent variety by
mixing them with heat-killed pathogenic bacteria.
 Transformation was demonstrated in 1944 by
Oswald Avery, Colin MacLeod, and Maclyn
McCarty, who showed gene transfer in
Streptococcus pneumoniae was pure DNA.
 Avery, Macleod and McCarty call the uptake and
incorporation of DNA by bacteria transformation.
Transformation - Mechanisms
 Bacteria
 transformation refers to a genetic change
brought about by picking up naked strands of
DNA and expressing it, and competence refers
to the state of being able to take up DNA.
 Two different forms of competence should be
distinguished, natural and artificial.
Transformation - Mechanisms
 Bacteria - Natural competence
 Some bacteria (around 1% of all species) are naturally
capable of taking up DNA. Such species carry sets of
genes specifying machinery for bringing DNA across
the cell's membrane or membranes.
 The evolutionary function of these genes is
controversial. Although most textbooks and
researchers have assumed that cells take up DNA to
acquire new versions of genes, a simpler explanation
that fits most of the observations is that cells take up
DNA mainly as a source of nucleotides, which can be
used directly or broken down and used for other
purposes
Transformation - Mechanisms
 Bacteria - Artificial competence
 Artificial competence is not encoded in the cell's
genes.
 It is induced by laboratory procedures in which cells
are passively made permeable to DNA, using
conditions that do not normally occur in nature.
 These procedures are comparatively easy and
simple, and are widely used to genetically engineer
bacteria.
 Artificially competent cells of standard bacterial
strains may also be purchased frozen, ready to
use.Common Strain of E. coli - DH5α (alpha)
Transformation - Mechanisms
 Bacteria - Artificial competence - Temperature
 Chilling cells in the presence of divalent cations
such as Ca2+ (in CaCl2) prepares the cell walls to
become permeable to plasmid DNA.
 Cells are incubated with the DNA and then briefly
heat shocked (42oC for 30-120 seconds), which
causes the DNA to enter the cell.
 This method works well for circular plasmid DNAs
but not for linear molecules such as fragments of
chromosomal DNA.
 An excellent preparation of competent cells will give
~108 colonies per μg of plasmid. A poor preparation
will be about 104/μg or less. Good non-commercial
preps should give 105 to 106 transformants per
microgram of plasmid.
Transformation - Mechanisms
What is a plasmid again?
 A plasmid DNA molecule contains sequences
allowing it to be replicated in the cell
independently of the chromosome.
 Plasmids used in experiments will usually
also contain an antibiotic resistance gene
which is placed in a bacterial strain that has
no antibiotic resistance.
 Therefore, only transformed bacteria will
grow on a media containing the antibiotic.
Transformation - Mechanisms
 Bacteria - Artificial competence –
Electroporation
 Electroporation is another way to make holes in
cells, by briefly shocking them with an electric
field of 100-200V/cm.
 Now plasmid DNA can enter the cell through
these holes.
 Natural membrane-repair mechanisms will close
these holes afterwards.
Transformation - Mechanisms
 Bacteria - Artificial competence – Lipofection
 Lipofection (or liposome transfection) is a
technique used to inject genetic material into a
cell by means of liposomes which are vesicles
that can easily merge with the cell membrane
since they are both made of a phospholipid
bilayer.
 The vescicle fuses with the cell membrane
(similar to how two oil spots at the top of a broth
will fuse) and the contents of the vesicle & the cell
are combined.
Transformation vs. Transfection
 Transfection (def): the introduction of
foreign material into eukaryotic cells.
 This typically involves opening transient
pores or 'holes' in the cell plasma
membrane, to allow uptake of material.
Transfection Mechanisms
Yeasts and Fungi
 These methods (and more) are currently known to
transform yeasts:
 Two-hybrid System Protocol:
 The two-hybrid system involve the use of two different plasmids in a
single yeast cell.
 One plasmid contains a cloned gene or DNA sequence of interest
while the other plasmid contains a library of genomic or cDNA.
(later)
 Frozen Yeast Protocol:
 Frozen yeast cells that are competent for transformation after
thawing.
 Gene Gun Transformation:
 Gold or tungsten nanoparticles can be shot at fungal cells growing
on PDA, transforming them.
 Protoplast Transformation:
 Fungal spores can be turned into protoplasts which can then be
soaked in DNA solution and transformed.
Transfection Mechanisms
 Plants - A number of mechanisms are available to transfer DNA
into an organism, these include:
 Agrobacterium is a genus of Gram-negative bacteria that
uses horizontal gene transfer to cause tumors in plants.
Agrobacterium tumefaciens is the most commonly studied
species in this genus.
 Horizontal gene transfer (HGT), also Lateral gene
transfer (LGT), is any process in which an organism
incorporates genetic material from another organism without
being the offspring of that organism. By contrast, vertical
transfer occurs when an organism receives genetic material
from its ancestor, e.g. its parent or a species from which it
evolved. Most thinking in genetics has focused upon vertical
transfer, but there is a growing awareness that horizontal
gene transfer is a highly significant phenomenon, and
amongst single-celled organisms perhaps the dominant
form of genetic transfer. Artificial horizontal gene transfer is
a form of genetic engineering.
Transfection Mechanisms
 Plants - A number of mechanisms are available to transfer
DNA into an organism, these include:
 Agrobacterium mediated transformation is the easiest
and most simple plant transformation. Plant tissue (often
leaves) are cut in small pieces, eg. 10x10mm, and
soaked for 10 minutes in a fluid containing suspended
agrobacterium. Some cells along the cut will be
transformed by the bacterium, that inserts its DNA into
the cell.
 Placed on selectable rooting and shooting media, the
plants will regrow. Some plants species can be
transformed just by dipping the flowers into suspension
of Agrobacteria and then planting the seeds in a
selective medium.
 Unfortunately, many plants are not transformable by this
method.
Transfection Mechanisms
 Plants
 Electroporation: make holes in cell walls using
electricity, that allows DNA to enter.
 Viral transformation: Package your genetic material into
a suitable plant virus and then use the modified virus
for infection of the plant.
 Genomes of most plant viruses consist of single
stranded RNA which replicates in the cytoplasm of
infected cell.
 So this method is not a real transformation (why?) …
since the inserted genes never reach the nucleus of the
cell and do not integrate into the host genome.
 The progeny of the infected plants is virus free and also
free of the inserted gene
Transfection Mechanisms
 Plants
 Particle bombardment (gene gun): Coat small gold
or tungsten particles with DNA and shoot them into
young plant cells or plant embryos. Some genetic
material will stay in the cells and transform them.
This method also allows transformation of plant
plastids.
 The transformation efficiency is lower than in agrobacterial
mediated transformation, but most plants can be
transformed with this method.
Transfection Mechanisms
 More on the “gene gun”
 The target of a gene gun is often a callus of undifferentiated
plant cells growing on gel medium in a petri dish. After the
gold particles have impacted the dish, the gel and callus are
largely disrupted. However, some cells were not obliterated
in the impact, and have successfully enveloped a DNA
coated tungsten particle, whose DNA eventually migrates to
and integrates into a plant chromosome.
 Cells from the entire petri dish can be re-collected and
selected for successful integration and expression of new
DNA using modern biochemical techniques
 Selected single cells from the callus can be treated with a
series of plant hormones, such as auxins and gibberellins,
and each may divide and differentiate into the organized,
specialized, tissue cells of an entire plant. This capability of
total re-generation is called totipotency. The new plant that
originated from a successfully shot cell may have new
genetic (heritable) traits.
Transfection Mechanisms
 Gene gun with Humans and Animals
 Gene guns have also been used to deliver DNA
vaccines to experimental animals.
Theoretically, it may be used in humans as well.
 The delivery of plasmids into rat neurons
through the use of a gene gun is also used as a
pharmacological precursor in studying the
effects of neurodegenerative diseases such as
Alzheimer's Disease.
 The Gene gun technique is also popularly used
in Edible vaccine production technique, where
the nano gold particles coated with plant gene
under the high vacuum pressurized chamber is
transformed into suitable plant tissues.
Transfection Mechanisms
 Animals
 Microinjection: use a thin needle and inject
the DNA directly in the core of embryonic
cells.
 Viral transformation: Package genetic
material into a virus, which delivers the
genetic material to target host cells.