Transformations of Cells (and Transfections too)

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Transformations of Cells (and Transfections too)

• Bacteria, Fungi, Plants, and Animal Cells
  Recombinant DNA Technology

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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

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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.

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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.

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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

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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 - DH5a
  (alpha)

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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

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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.

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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.

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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.

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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.