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Transformations of Cells (and Transfections too)

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Title: Transformations of Cells (and Transfections too)


1
Transformations of Cells (and Transfections too)
  • Bacteria, Fungi, Plants, and Animal Cells
    Recombinant DNA Technology

2
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

3
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.

4
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.

5
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

6
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)

7
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.

8
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.

9
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.

10
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.

11
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.

12
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.

13
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.

14
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.

15
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

16
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.

17
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.

18
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.

19
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.
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