Transformations of Transformations of Cells Cells (and Transfections too)(and Transfections too)

Bacteria, Fungi, Plants, and Animal Bacteria, Fungi, Plants, and Animal Cells & Recombinant DNA Cells & Recombinant DNA TechnologyTechnology

TransformationTransformation TransformationTransformation (def): (def): the genetic alteration of the genetic alteration of

a cell resulting from the introduction, uptake a cell resulting from the introduction, uptake and expression of foreign genetic material and expression of foreign genetic material (DNA) in molecular biology(DNA) in molecular biology

This can be done to Bacteria, Fungi, Plants, This can be done to Bacteria, Fungi, Plants, and Animal cellsand Animal cells

Transformation - HistoryTransformation - History 1928 - Frederick Griffith transforms nonpathogenic 1928 - Frederick Griffith transforms nonpathogenic

pneumococcus bacteria into a virulent variety by pneumococcus bacteria into a virulent variety by mixing them with heat-killed pathogenic bacteria. mixing them with heat-killed pathogenic bacteria.

Transformation was demonstrated in 1944 by Transformation was demonstrated in 1944 by Oswald Avery, Colin MacLeod, and Maclyn Oswald Avery, Colin MacLeod, and Maclyn McCarty, who showed gene transfer in McCarty, who showed gene transfer in Streptococcus pneumoniae Streptococcus pneumoniae was pure DNA. was pure DNA.

Avery, Macleod and McCarty call the uptake and Avery, Macleod and McCarty call the uptake and incorporation of DNA by bacteria transformation.incorporation of DNA by bacteria transformation.

Transformation - MechanismsTransformation - Mechanisms BacteriaBacteria

transformationtransformation refers to a genetic change refers to a genetic change brought about by picking up naked strands of brought about by picking up naked strands of DNA and expressing it, and DNA and expressing it, and competence competence refers refers to the state of being able to take up DNA. to the state of being able to take up DNA.

Two different forms of competence should be Two different forms of competence should be distinguished, natural and artificial.distinguished, natural and artificial.

Transformation - MechanismsTransformation - Mechanisms Bacteria - Bacteria - Natural competenceNatural competence

Some bacteria (around 1% of all species) are naturally Some bacteria (around 1% of all species) are naturally capable of taking up DNA. Such species carry sets of capable of taking up DNA. Such species carry sets of genes specifying machinery for bringing DNA across genes specifying machinery for bringing DNA across the cell's membrane or membranes. the cell's membrane or membranes.

The evolutionary function of these genes is The evolutionary function of these genes is controversial. Although most textbooks and controversial. Although most textbooks and researchers have assumed that cells take up DNA to researchers have assumed that cells take up DNA to acquire new versions of genes, a simpler explanation acquire new versions of genes, a simpler explanation that fits most of the observations is that cells take up that fits most of the observations is that cells take up DNA mainly as a source of nucleotides, which can be DNA mainly as a source of nucleotides, which can be used directly or broken down and used for other used directly or broken down and used for other purposespurposes

Transformation - MechanismsTransformation - Mechanisms

Bacteria - Bacteria - Artificial competenceArtificial competence Artificial competence is not encoded in the cell's Artificial competence is not encoded in the cell's

genes. genes. It is induced by laboratory procedures in which cells It is induced by laboratory procedures in which cells

are passively made permeable to DNA, using are passively made permeable to DNA, using conditions that do not normally occur in nature. conditions that do not normally occur in nature.

These procedures are comparatively easy and These procedures are comparatively easy and simple, and are widely used to genetically engineer simple, and are widely used to genetically engineer bacteria. bacteria.

Artificially competent cells of standard bacterial Artificially competent cells of standard bacterial strains may also be purchased frozen, ready to strains may also be purchased frozen, ready to use.Common Strain of use.Common Strain of E. coli - E. coli - DH5DH5αα (alpha) (alpha)

Transformation - MechanismsTransformation - Mechanisms Bacteria - Bacteria - Artificial competence - TemperatureArtificial competence - Temperature

Chilling cells in the presence of divalent cations Chilling cells in the presence of divalent cations such as Ca2+ (in CaClsuch as Ca2+ (in CaCl22) prepares the cell walls to ) prepares the cell walls to become permeable to plasmid DNA. become permeable to plasmid DNA.

Cells are incubated with the DNA and then briefly Cells are incubated with the DNA and then briefly heat shocked (42heat shocked (42ooC for 30-120 seconds), which C for 30-120 seconds), which causes the DNA to enter the cell. causes the DNA to enter the cell.

This method works well for circular plasmid DNAs This method works well for circular plasmid DNAs but not for linear molecules such as fragments of but not for linear molecules such as fragments of chromosomal DNA. chromosomal DNA.

An excellent preparation of competent cells will give An excellent preparation of competent cells will give ~10~1088 colonies per μg of plasmid. A poor preparation colonies per μg of plasmid. A poor preparation will be about 10will be about 1044/μg or less. Good non-commercial /μg or less. Good non-commercial preps should give 10preps should give 105 5 to 10to 1066 transformants per transformants per microgram of plasmid.microgram of plasmid.

Transformation - MechanismsTransformation - MechanismsWhat is a plasmid again?What is a plasmid again? A plasmid DNA molecule contains A plasmid DNA molecule contains

sequences allowing it to be replicated in the sequences allowing it to be replicated in the cell independently of the chromosome. cell independently of the chromosome.

Plasmids used in experiments will usually Plasmids used in experiments will usually also contain an antibiotic resistance gene also contain an antibiotic resistance gene which is placed in a bacterial strain that has which is placed in a bacterial strain that has no antibiotic resistance. no antibiotic resistance.

Therefore, only transformed bacteria will Therefore, only transformed bacteria will grow on a media containing the antibiotic.grow on a media containing the antibiotic.

Transformation - MechanismsTransformation - Mechanisms

Bacteria - Bacteria - Artificial competence – Artificial competence – ElectroporationElectroporation Electroporation is another way to make holes in Electroporation is another way to make holes in

cells, by briefly shocking them with an electric cells, by briefly shocking them with an electric field of 100-200V/cm. field of 100-200V/cm.

Now plasmid DNA can enter the cell through Now plasmid DNA can enter the cell through these holes. these holes.

Natural membrane-repair mechanisms will close Natural membrane-repair mechanisms will close these holes afterwards.these holes afterwards.

Transformation - MechanismsTransformation - Mechanisms

Bacteria - Bacteria - Artificial competence – LipofectionArtificial competence – Lipofection LipofectionLipofection (or liposome transfection) is a technique (or liposome transfection) is a technique

used to inject genetic material into a cell by means of used to inject genetic material into a cell by means of liposomes which are vesicles that can easily merge liposomes which are vesicles that can easily merge with the cell membrane since they are both made of a with the cell membrane since they are both made of a phospholipid bilayer.phospholipid bilayer.

The vescicle fuses with the cell membrane (similar to The vescicle fuses with the cell membrane (similar to how two oil spots at the top of a broth will fuse) and how two oil spots at the top of a broth will fuse) and the contents of the vesicle & the cell are combinedthe contents of the vesicle & the cell are combined..

Transformation vs. TransfectionTransformation vs. Transfection

TransfectionTransfection (def): the introduction of (def): the introduction of foreign material into eukaryotic cells. foreign material into eukaryotic cells.

This typically involves opening transient This typically involves opening transient pores or 'holes' in the cell plasma pores or 'holes' in the cell plasma membrane, to allow uptake of material. membrane, to allow uptake of material.

Transfection MechanismsTransfection MechanismsYeasts and FungiYeasts and Fungi These methods (and more) are currently known to These methods (and more) are currently known to

transform yeasts: transform yeasts: Two-hybrid System Protocol:Two-hybrid System Protocol:

The two-hybrid system involve the use of two different plasmids in a The two-hybrid system involve the use of two different plasmids in a single yeast cell. single yeast cell.

One plasmid contains a cloned gene or DNA sequence of interest One plasmid contains a cloned gene or DNA sequence of interest while the other plasmid contains a library of genomic or cDNA. while the other plasmid contains a library of genomic or cDNA. (later)(later)

Frozen Yeast Protocol:Frozen Yeast Protocol: Frozen yeast cells that are competent for transformation after Frozen yeast cells that are competent for transformation after

thawing. thawing. Gene Gun Transformation:Gene Gun Transformation:

Gold or tungsten nanoparticles can be shot at fungal cells growing Gold or tungsten nanoparticles can be shot at fungal cells growing on PDA, transforming them. on PDA, transforming them.

Protoplast Transformation:Protoplast Transformation: Fungal spores can be turned into protoplasts which can then be Fungal spores can be turned into protoplasts which can then be

soaked in DNA solution and transformed. soaked in DNA solution and transformed.

Transfection MechanismsTransfection Mechanisms Plants - Plants - A number of mechanisms are available to transfer DNA A number of mechanisms are available to transfer DNA

into an organism, these include:into an organism, these include: AgrobacteriumAgrobacterium is a is a genusgenus of of Gram-negativeGram-negative bacteriabacteria that that

uses horizontal gene transfer to cause tumors in plants. uses horizontal gene transfer to cause tumors in plants. Agrobacterium tumefaciensAgrobacterium tumefaciens is the most commonly studied is the most commonly studied species in this genus. species in this genus.

Horizontal gene transfer (HGT)Horizontal gene transfer (HGT), also , also Lateral gene Lateral gene transfer (LGT)transfer (LGT), is any process in which an organism , is any process in which an organism incorporates genetic material from another organism without incorporates genetic material from another organism without being the offspring of that organism. By contrast, being the offspring of that organism. By contrast, vertical vertical transfertransfer occurs when an organism receives genetic material occurs when an organism receives genetic material from its ancestor, e.g. its parent or a species from which it from its ancestor, e.g. its parent or a species from which it evolved. Most thinking in genetics has focused upon vertical evolved. Most thinking in genetics has focused upon vertical transfer, but there is a growing awareness that horizontal transfer, but there is a growing awareness that horizontal gene transfer is a highly significant phenomenon, and gene transfer is a highly significant phenomenon, and amongst single-celled organisms perhaps the dominant amongst single-celled organisms perhaps the dominant form of genetic transfer. Artificial horizontal gene transfer is form of genetic transfer. Artificial horizontal gene transfer is a form of genetic engineering. a form of genetic engineering.

Transfection MechanismsTransfection Mechanisms Plants - Plants - A number of mechanisms are available to transfer A number of mechanisms are available to transfer

DNA into an organism, these include:DNA into an organism, these include: AgrobacteriumAgrobacterium mediated transformation is the easiest mediated transformation is the easiest

and most simple plant transformation. Plant tissue (often and most simple plant transformation. Plant tissue (often leaves) are cut in small pieces, eg. 10x10mm, and leaves) are cut in small pieces, eg. 10x10mm, and soaked for 10 minutes in a fluid containing suspended soaked for 10 minutes in a fluid containing suspended agrobacterium. Some cells along the cut will be agrobacterium. Some cells along the cut will be transformed by the bacterium, that inserts its DNA into transformed by the bacterium, that inserts its DNA into the cell.the cell.

Placed on selectable rooting and shooting media, the Placed on selectable rooting and shooting media, the plants will regrow. Some plants species can be plants will regrow. Some plants species can be transformed just by dipping the flowers into suspension transformed just by dipping the flowers into suspension of Agrobacteria and then planting the seeds in a of Agrobacteria and then planting the seeds in a selective medium. selective medium.

Unfortunately, many plants are not transformable by this Unfortunately, many plants are not transformable by this method. method.

Transfection MechanismsTransfection Mechanisms PlantsPlants

ElectroporationElectroporation: make holes in cell walls using : make holes in cell walls using electricity, that allows DNA to enter. electricity, that allows DNA to enter.

Viral transformationViral transformation: Package your genetic material into a : Package your genetic material into a suitable plant virus and then use the modified virus for suitable plant virus and then use the modified virus for infection of the plant. infection of the plant.

Genomes of most plant viruses consist of single Genomes of most plant viruses consist of single stranded RNA which replicates in the cytoplasm of stranded RNA which replicates in the cytoplasm of infected cell. infected cell.

So this method is not a real transformation So this method is not a real transformation (why?(why?) … ) … since the inserted genes never reach the nucleus of the since the inserted genes never reach the nucleus of the cell and do not integrate into the host genome. cell and do not integrate into the host genome.

The progeny of the infected plants is virus free and also The progeny of the infected plants is virus free and also free of the inserted genefree of the inserted gene

Transfection MechanismsTransfection Mechanisms PlantsPlants

Particle bombardment (gene gun): Coat small gold Particle bombardment (gene gun): Coat small gold or tungsten particles with DNA and shoot them into or tungsten particles with DNA and shoot them into young plant cells or plant embryos. Some genetic young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. material will stay in the cells and transform them. This method also allows transformation of plant This method also allows transformation of plant plastids. plastids. The transformation efficiency is lower than in agrobacterial The transformation efficiency is lower than in agrobacterial

mediated transformation, but most plants can be mediated transformation, but most plants can be transformed with this method. transformed with this method.

Transfection MechanismsTransfection Mechanisms More on the “gene gun”More on the “gene gun” The target of a gene gun is often a The target of a gene gun is often a calluscallus of undifferentiated of undifferentiated

plant cells growing on gel medium in a plant cells growing on gel medium in a petripetri dish dish. After the . After the gold particles have impacted the dish, the gel and callus are gold particles have impacted the dish, the gel and callus are largely disrupted. However, some cells were not obliterated largely disrupted. However, some cells were not obliterated in the impact, and have successfully enveloped a DNA in the impact, and have successfully enveloped a DNA coated tungsten particle, whose DNA eventually migrates to coated tungsten particle, whose DNA eventually migrates to and integrates into a plant and integrates into a plant chromosomechromosome..

Cells from the entire petri dish can be re-collected and Cells from the entire petri dish can be re-collected and selected for successful integration and expression of new selected for successful integration and expression of new DNA using modern biochemical techniquesDNA using modern biochemical techniques

Selected single cells from the callus can be treated with a Selected single cells from the callus can be treated with a series of plant hormones, such as series of plant hormones, such as auxinsauxins and and gibberellinsgibberellins, , and each may divide and differentiate into the organized, and each may divide and differentiate into the organized, specialized, tissue cells of an entire plant. This capability of specialized, tissue cells of an entire plant. This capability of total re-generation is called total re-generation is called totipotencytotipotency. The new plant that . The new plant that originated from a successfully shot cell may have new originated from a successfully shot cell may have new genetic (heritable) traits.genetic (heritable) traits.

Transfection MechanismsTransfection Mechanisms Gene gun with Humans and AnimalsGene gun with Humans and Animals Gene guns have also been used to deliver Gene guns have also been used to deliver

DNA vaccinesDNA vaccines to experimental animals. to experimental animals. Theoretically, it may be used in humans as Theoretically, it may be used in humans as well.well.

The delivery of plasmids into rat neurons The delivery of plasmids into rat neurons through the use of a gene gun is also used as a through the use of a gene gun is also used as a pharmacological precursor in studying the pharmacological precursor in studying the effects of neurodegenerative diseases such as effects of neurodegenerative diseases such as Alzheimer's DiseaseAlzheimer's Disease..

The Gene gun technique is also popularly used The Gene gun technique is also popularly used in Edible vaccine production technique, where in Edible vaccine production technique, where the nano gold particles coated with plant gene the nano gold particles coated with plant gene under the high vacuum pressurized chamber is under the high vacuum pressurized chamber is transformed into suitable plant tissues.transformed into suitable plant tissues.

Transfection MechanismsTransfection Mechanisms AnimalsAnimals

MicroinjectionMicroinjection: use a thin needle and inject the : use a thin needle and inject the DNA directly in the core of embryonic cells. DNA directly in the core of embryonic cells.

Viral transformationViral transformation: Package genetic material : Package genetic material into a virus, which delivers the genetic into a virus, which delivers the genetic material to target host cells.material to target host cells.