M.sc.Micro 3rd Sem Unit 3

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M.Sc.Micro R- DNA Technology Unit 3

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Transformation

In molecular biology,transformation is genetic alteration of a cell resulting from the direct uptake,

incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and

taken up through the cell membrane(s). Transformation occurs naturally in some species of bacteria, but

it can also be affected by artificial means in other cells. For transformation to happen, bacteria must be

in a state ofcompetence, which might occur as a time-limited response to environmental conditions

such as starvation and cell density.

Transformation is one of three processes by which exogenous genetic material may be introduced into a

bacterial cell, the other two being conjugation (transfer of genetic material between two bacterial cells

in direct contact) and transduction (injection of foreign DNA by a bacteriophage virus into the host

bacterium).

"Transformation" may also be used to describe the insertion of new genetic material into nonbacterial

cells, including animal and plant cells; however, because "transformation" has a special meaning in

relation to animal cells, indicating progression to a cancerous state, the term should be avoided for

animal cells when describing introduction of exogenous genetic material. Introduction of foreign DNA

into eukaryotic cells is often called "transfection

History

Transformation was first demonstrated in 1928 by British bacteriologist Frederick Griffith. Griffith

discovered that a harmless strain ofStreptococcus pneumoniaecould be made virulent after being

exposed to heat-killed virulent strains. Griffith hypothesized that some "transforming principle" fromthe heat-killed strain was responsible for making the harmless strain virulent. In 1944 this "transforming

principle" was identified as being genetic by Oswald Avery, Colin MacLeod, and Maclyn McCarty. They

isolated DNA from a virulent strain ofS. pneumoniae and using just this DNA were able to make a

harmless strain virulent. They called this uptake and incorporation of DNA by bacteria "transformation"

(See Avery-MacLeod-McCarty experiment). The results of Avery et al.'s experiments were at first

skeptically received by the scientific community and it was not until the development ofgenetic

markers and the discovery of other methods of genetic transfer (conjugation in 1947 and transduction in

1953) by Joshua Lederberg that Avery's experiments were accepted.

It was originally thought thatEscherichia coli, a commonly used laboratory organism, was refractory to

transformation. However, in 1970, Morton Mandel and Akiko Higa showed that E. colimay be induced

to take up DNA frombacteriophage without the use ofhelper phage after treatment with calcium

chloride solution. Two years later in 1972, Stanley Cohen, Annie Chang and Leslie Hsu showed that CaCl

2 treatment is also effective for transformation of plasmid DNA ]The method of transformation by
http://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Introduction_to_geneticshttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Gene_expressionhttp://en.wikipedia.org/wiki/Genehttp://en.wikipedia.org/wiki/Exogenous_DNAhttp://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Competence_(biology)http://en.wikipedia.org/wiki/Bacterial_conjugationhttp://en.wikipedia.org/wiki/Transduction_(genetics)http://en.wikipedia.org/wiki/Eukaryotic_cellhttp://en.wikipedia.org/wiki/Transfectionhttp://en.wikipedia.org/wiki/Frederick_Griffithhttp://en.wikipedia.org/wiki/Streptococcus_pneumoniaehttp://en.wikipedia.org/wiki/Streptococcus_pneumoniaehttp://en.wikipedia.org/wiki/Streptococcus_pneumoniaehttp://en.wikipedia.org/wiki/Oswald_Averyhttp://en.wikipedia.org/wiki/Colin_Munro_MacLeodhttp://en.wikipedia.org/wiki/Maclyn_McCartyhttp://en.wikipedia.org/wiki/Avery-MacLeod-McCarty_experimenthttp://en.wikipedia.org/wiki/Genetic_markershttp://en.wikipedia.org/wiki/Genetic_markershttp://en.wikipedia.org/wiki/Bacterial_conjugationhttp://en.wikipedia.org/wiki/Transduction_(genetics)http://en.wikipedia.org/wiki/Joshua_Lederberghttp://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Lambda_phagehttp://en.wikipedia.org/wiki/Lambda_phagehttp://en.wikipedia.org/wiki/Lambda_phagehttp://en.wikipedia.org/wiki/Helper_virushttp://en.wikipedia.org/wiki/Stanley_Cohen_(biochemist)http://en.wikipedia.org/wiki/Transformation_(genetics)#cite_note-4http://en.wikipedia.org/wiki/Transformation_(genetics)#cite_note-4http://en.wikipedia.org/wiki/Transformation_(genetics)#cite_note-4http://en.wikipedia.org/wiki/Stanley_Cohen_(biochemist)http://en.wikipedia.org/wiki/Helper_virushttp://en.wikipedia.org/wiki/Lambda_phagehttp://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Joshua_Lederberghttp://en.wikipedia.org/wiki/Transduction_(genetics)http://en.wikipedia.org/wiki/Bacterial_conjugationhttp://en.wikipedia.org/wiki/Genetic_markershttp://en.wikipedia.org/wiki/Genetic_markershttp://en.wikipedia.org/wiki/Avery-MacLeod-McCarty_experimenthttp://en.wikipedia.org/wiki/Maclyn_McCartyhttp://en.wikipedia.org/wiki/Colin_Munro_MacLeodhttp://en.wikipedia.org/wiki/Oswald_Averyhttp://en.wikipedia.org/wiki/Streptococcus_pneumoniaehttp://en.wikipedia.org/wiki/Frederick_Griffithhttp://en.wikipedia.org/wiki/Transfectionhttp://en.wikipedia.org/wiki/Eukaryotic_cellhttp://en.wikipedia.org/wiki/Transduction_(genetics)http://en.wikipedia.org/wiki/Bacterial_conjugationhttp://en.wikipedia.org/wiki/Competence_(biology)http://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Exogenous_DNAhttp://en.wikipedia.org/wiki/Genehttp://en.wikipedia.org/wiki/Gene_expressionhttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Introduction_to_geneticshttp://en.wikipedia.org/wiki/Molecular_biology


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Mandel and Higa was later improved upon by Douglas Hanahan. The discovery of artificially induced

competence in E. colicreated an efficient and convenient procedure for transforming bacteria which

allows for simpler molecular cloning methods in biotechnology and research, and it is now a routinely

used laboratory procedure.

Transformation using electroporation was developed in the late 1980s, increasing the efficiency of in-

vitro transformation and increasing the number ofbacterial strains that could be

transformed. Transformation of animal and plant cells was also investigated with the first transgenic

mouse being created by injecting a gene for a rat growth hormone into a mouse embryo in 1982. In

1907 a bacterium that caused plant tumors,Agrobacterium tumefaciens, was discovered and in the

early 1970s the tumor inducing agent was found to be a DNA plasmid called the Ti plasmid. By removing

the genes in the plasmid that caused the tumor and adding in novel genes researchers were able to

infect plants withA. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the

plants. Not all plant cells are susceptible to infection byA. tumefaciens so other methods weredeveloped including electroporation and micro-injection. Particle bombardment was made possible with

the invention of the Biolistic Particle Delivery System (gene gun) by John Sanford in the 1980s.

Methods and mechanisms

Bacteria

Bacterial transformation may be referred to as a stable genetic change brought about by the uptake

ofnaked DNA (DNA without associated cells or proteins) and competence refers to the state of being

able to take up exogenous DNA from the environment. There are two forms of transformation and

competence: natural and artificial.

Natural transformation

Natural transformation is a bacterial adaptation for DNA transfer that depends on the expression of

numerous bacterial genes whose products appear to be designed to carry out this process. In general,

transformation is a complex, energy requiring developmental process. In order for a bacterium to bind,

take up and recombine exogenous DNA into its chromosome it must become competent, that is, enter a

special physiological state. Competence development in Bacillus subtilis requires expression of about 40

genes. The DNA integrated into the host chromosome is usually (but with rare exceptions) derived fromanother bacterium of the same species, and is thus homologous to the resident chromosome.

In B. subtilis the length of the transferred DNA is greater than 1271 kb (more than 1 million bases). The

length transferred is likely double stranded DNA and is often more than a third of the total chromosome

length of 4215 kb. It appears that about 7-9% of the recipient cells take up an entire chromosome.
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The capacity for natural transformation appears to occur in a number of prokaryotes, and thus far 67

prokaryotic species (in seven different phyla) are known to undergo this process.

Competence for transformation is typically induced by high cell density and/or nutritional limitation,

conditions associated with the stationary phase of bacterial growth. Transformation in Haemophilus

influenzae occurs most efficiently at the end of exponential growth as bacterial growth approaches

stationary phase. Transformation in Streptococcus mutans, as well as in many other streptococci,

occurs at high cell density and is associated with biofilm formation. Competence in B. subtilis is induced

toward the end of logarithmic growth, especially under conditions of amino acid limitation.

Transformation, as an adaptation for DNA repair

Competence is specifically induced by DNA damaging conditions. For instance, transformation is induced

in Streptococcus pneumoniae by the DNA damaging agents mitomycin C (a DNA crosslinking agent) and

fluoroquinolone (a topoisomerase inhibitor that causes double-strand breaks) In B. subtilis,

transformation is increased by UV light, a DNA damaging agent. In Helicobacter pylori, ciprofloxacin,

which interacts with DNA gyrase and introduces double-strand breaks, induces expression of

competence genes, thus enhancing the frequency of transformatio[ Using Legionella pneumophila,

Charpentier et al. tested 64 toxic molecules to determine which of these induce competence. Of these,

only six, all DNA damaging agents caused strong induction. These DNA damaging agents were mitomycin

C (which causes DNA inter-strand crosslinks), norfloxacin, ofloxacin and nalidixic acid (inhibitors of DNA

gyrase that cause double-strand breaks , bicyclomycin (causes single- and double-strand breaks, and

hydroxyurea (induces DNA base oxidation). UV light also induced competence in L. pneumophila.

Charpentier et al. suggested that competence for transformation probably evolved as a DNA damageresponse.

Logarithmically growing bacteria differ from stationary phase bacteria with respect to the number of

genome copies present in the cell, and this has implications for the capability to carry out an important

DNA repair process. During logarithmic growth, two or more copies of any particular region of the

chromosome may be present in a bacterial cell, as cell division is not precisely matched with

chromosome replication. The process of homologous recombinational repair (HRR) is a key DNA repair

process that is especially effective for repairing double-strand damages, such as double-strand breaks.

This process depends on a second homologous chromosome in addition to the damaged chromosome.During logarithmic growth, a DNA damage in one chromosome may be repaired by HRR using sequence

information from the other homologous chromosome. Once cells approach stationary phase, however,

they typically have just one copy of the chromosome, and HRR requires input of homologous template

from outside the cell by transformation.
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To test whether the adaptive function of transformation is repair of DNA damages, a series of

experiments were carried out using B. subtilis irradiated by UV light as the damaging agent (reviewed by

Michod et al. and Bernstein et al.]) The results of these experiments indicated that transforming DNA

acts to repair potentially lethal DNA damages introduced by UV light in the recipient DNA. The particular

process responsible for repair was likely HRR. Transformation in bacteria can be viewed as a primitive

sexual process, since it involves interaction of homologous DNA from two individuals to form

recombinant DNA that is passed on to succeeding generations. Bacterial transformation in prokaryotes

may have been the ancestral process that gave rise to meiotic sexual reproduction in eukaryotes

Natural competence

About 1% of bacterial species are capable of naturally taking up DNA under laboratory conditions; more

may be able to take it up in their natural environments. DNA material can be transferred between

different strains of bacteria, in a process that is called horizontal gene transfer. Some species upon cell

death release their DNA to be taken up by other cells, however transformation works best with DNA

from closely related species.

These naturally competent bacteria carry sets of genes that provide the protein machinery to bring DNA

across the cell membrane(s). The transport of the exogeneous DNA into the cells may require proteins

that are involved in the assembly oftype IV pili and type II secretion system, as well as

DNA translocase complex at the cytoplasmic membrane. Due to the differences in structure of the cell

envelope between Gram-positive and Gram-negative bacteria, there are some differences in the

mechanisms of DNA uptake in these cells, however most of them share common features that involve

related proteins.

The DNA first binds to the surface of the competent cells on a DNA receptor, and passes through

the cytoplasmic membrane via DNA translocase. Only single-stranded DNA may pass through, one

strand is therefore degraded by nucleases in the process, and the translocated single-stranded DNA may

then be integrated into the bacterial chromosomes by a RecA-dependent process. In Gram-negative

cells, due to the presence of an extra membrane, the DNA requires the presence of a channel formed by

secretins on the outer membrane. Pilin may be required for competence however its role is

uncertain.]The uptake of DNA is generally non-sequence specific, although in some species the presence

of specific DNA uptake sequences may facilitate efficient DNA uptake.

Plants

A number of methods are available to transfer DNA into plant cells:

Agrobacteriummediated transformation is the easiest and most simple plant transformation. Planttissue (often leaves) are cut into small pieces, e.g. 10x10mm, and soaked for 10 minutes in a fluid
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containing suspendedAgrobacterium. 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 ofAgrobacterium and then planting the seeds in a selective medium. Unfortunately,

many plants are not transformable by this method.

Gene gun: Also referred to as particle bombardment, microprojectile bombardment, or biolistics.Particles of gold or tungsten are coated with DNA and then shot 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

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

Electroporation: make transient holes in cell membranes using electric shock; this allows DNA toenter as described above for Bacteria.

Viral transformation (transduction): Package the desired genetic material into a suitable plant virusand allow this modified virus to infect the plant. If the genetic material is DNA, it can recombine

with the chromosomes to produce transformant cells. However genomes of most plant viruses

consist of single stranded RNA which replicates in the cytoplasm of infected cell. For such genomes

this method is a form oftransfection and not a real transformation, 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.

Animals

Introduction of DNA into animal cells is usually called transfection, and is discussed in the correspondingarticle.
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Transfection ions

Transfection is the process of deliberately introducing nucleic acids into cells. The term is oftenused for non-viral methods in eukaryotic cells. It may also refer to other methods and cell types,although other terms are preferred: "transformation" is more often used to describe non-viral

DNA transfer in bacteria, non-animal eukaryotic cells and plant cells a distinctive sense oftransformation refers to spontaneous genetic modifications (mutations to cancerous cells(carcinogenesis), or under stress (UV irradiation)). Transduction is often used to describe virus-mediated DNA transfer. The word transfection is a blend oftrans- and infection.

Genetic material (such as supercoiled plasmid DNA orsiRNA constructs), or even proteins suchas antibodies, may be transfected.

Transfection of animal cells typically involves opening transient pores or "holes" in the cellmembrane, to allow the uptake of material. Transfection can be carried out using calciumphosphate, by electroporation, or by mixing a cationic lipid with the material to produce

liposomes, which fuse with the cell membrane and deposit their cargo inside.

Transfection can result in unexpected morphologies and abnormalities in target cells.

Terminology

The meaning of the term has evolved. The original meaning of transfection was "infection bytransformation," i.e., introduction of DNA (or RNA) from a prokaryote-infecting virus orbacteriophage into cells, resulting in an infection. Because the term transformation had anothersense in animal cell biology (a genetic change allowing long-term propagation in culture, oracquisition of properties typical of cancer cells), the term transfection acquired, for animal cells,

its present meaning of a change in cell properties caused by introduction of DNA.

Methods

There are various methods of introducing foreign DNA into a eukaryotic cell: some rely onphysical treatment (electroporation, nanoparticles, magnetofection), other on chemical materialsor biological particles (viruses) that are used as carriers.

Chemical-based transfection

Chemical-based transfection can be divided into several kinds: cyclodextrin, polymers,liposomes, or nanoparticles (with or without chemical or viral functionalization. See below).

One of the cheapest methods uses calcium phosphate, originally discovered by F. L. Graham andA. J. van der Eb in 1973. HEPES-buffered saline solution (HeBS) containing phosphate ions iscombined with a calcium chloride solution containing the DNA to be transfected. When the twoare combined, a fine precipitate of the positively charged calcium and the negatively chargedphosphate will form, binding the DNA to be transfected on its surface. The suspension of the
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precipitate is then added to the cells to be transfected (usually a cell culture grown in amonolayer). By a process not entirely understood, the cells take up some of the precipitate, andwith it, the DNA.

Other methods use highly branched organic compounds, so-called dendrimers, to bind the DNAand get it into the cell.

A very efficient method is the inclusion of the DNA to be transfected in liposomes, i.e. small,membrane-bounded bodies that are in some ways similar to the structure of a cell and canactually fuse with the cell membrane, releasing the DNA into the cell. For eukaryotic cells,transfection is better achieved using cationic liposomes (or mixtures), because the cells aremore sensitive. See lipofection for more details.

Another method is the use ofcationic polymers such as DEAE-dextran or polyethylenimine. Thenegatively charged DNA binds to the polycation and the complex is taken up by the cell viaendocytosis.

Non chemical methods

Electroporation (Gene electrotransfer) is a popular method, where transient increase in thepermeability of cell membrane is achieved when the cells are exposed to short pulses of anintense electric field.

Similarly, transfection applying sonic forces to cells, referred as Sono-poration. Optical transfection is a method where a tiny (~1 m diameter) hole is transiently generated in

the plasma membrane of a cell using a highly focused laser. This technique was first described in1984 by Tsukakoshi et al., who used a frequency tripled Nd:YAG to generate stable and transient

transfection of normal rat kidney cells.]In this technique, one cell at a time is treated, making itparticularly useful for single cell analysis.

Protoplast fusion is a technique in which transformed bacterial cells are treated with lysozymein order to remove the cell wall. Following this, fusogenic agents (e.g., Sendai virus, PEG, orelectroporation)are used in order to fuse the protoplast carrying the gene of interest with thetarget recipient cell. A major disadvantage of this method is that bacterial components are non-specifically introduced into the target cell as well.

Impalefection is a method of introducing DNA bound to a surface of a nanofiber that is insertedinto a cell. This approach can also be implemented with arrays of nanofibers that are introduced

into large numbers of cells and intact tissue.

Hydrodynamic delivery In mice and rats, but to a lesser extent in larger animals, DNA most oftenin plasmids, including transposons, can be delivered to the liver using hydrodynamic injectionthat involves infusion of a relatively large volume in the blood in less than 10 seconds; nearly allof the DNA is expressed in the liver by this procedure.
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Particle-based methods

A direct approach to transfection is the gene gun, where the DNA is coupled to a nanoparticle ofan inert solid (commonly gold) which is then "shot" directly into the target cell's nucleus.

Magnetofection, or Magnet assisted transfection is a transfection method, which uses magneticforce to deliver DNA into target cells. Nucleic acids are first associated with magneticnanoparticles. Then, application of magnetic force drives the nucleic acid particle complexestowards and into the target cells, where the cargo is released.

Impalefection is carried out by impaling cells by elongated nanostructures and arrays of suchnanostructures such as carbon nanofibers or silicon nanowires which have been functionalizedwith plasmid DNA.

Viral methods

DNA can also be introduced into cells using viruses as a carrier. In such cases, the technique iscalled viral transduction, and the cells are said to be transduced.

Other (and hybrid) methods

Other methods of transfection include nucleofection, heat shock.

Stable and transient transfection

For some applications of transfection, it is sufficient if the transfected genetic material is onlytransiently expressed. Since the DNA introduced in the transfection process is usually not

integrated into the nuclear genome, the foreign DNA will be diluted through mitosis or degraded.Cell lines expressing the EpsteinBarr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40large-T antigen, allow episomal amplification of plasmids containing the viral EBV (293E) orSV40 (293T) origins of replication, greatly reducing the rate of dilution.

If it is desired that the transfected gene actually remain in the genome of the cell and its daughtercells, a stable transfection must occur. To accomplish this, a marker gene is co-transfected,which gives the cell some selectable advantage, such as resistance towards a certain toxin. Some(very few) of the transfected cells will, by chance, have integrated the foreign genetic materialinto their genome. If the toxin is then added to the cell culture, only those few cells with themarker gene integrated into their genomes will be able to proliferate, while other cells will die.

After applying this selective stress (selection pressure) for some time, only the cells with a stabletransfection remain and can be cultivated further.

A common agent for selecting stable transfection is Geneticin, also known as G418, which is atoxin that can be neutralized by the product of the neomycin resistance gene.
http://en.wikipedia.org/wiki/Gene_gunhttp://en.wikipedia.org/wiki/Nanoparticlehttp://en.wikipedia.org/wiki/Inerthttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Magnetofectionhttp://en.wikipedia.org/wiki/Magnet_assisted_transfectionhttp://en.wikipedia.org/wiki/Impalefectionhttp://en.wikipedia.org/wiki/Carbon_nanofibershttp://en.wikipedia.org/wiki/Siliconhttp://en.wikipedia.org/wiki/Nanowireshttp://en.wikipedia.org/wiki/Plasmidhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Virushttp://en.wikipedia.org/wiki/Transduction_%28genetics%29http://en.wikipedia.org/wiki/Nucleofectionhttp://en.wikipedia.org/wiki/Heat_shockhttp://en.wikipedia.org/wiki/Mitosishttp://en.wikipedia.org/wiki/Marker_genehttp://en.wikipedia.org/wiki/Toxinhttp://en.wikipedia.org/wiki/Cell_proliferationhttp://en.wikipedia.org/wiki/Geneticinhttp://en.wikipedia.org/wiki/Neomycinhttp://en.wikipedia.org/wiki/Neomycinhttp://en.wikipedia.org/wiki/Geneticinhttp://en.wikipedia.org/wiki/Cell_proliferationhttp://en.wikipedia.org/wiki/Toxinhttp://en.wikipedia.org/wiki/Marker_genehttp://en.wikipedia.org/wiki/Mitosishttp://en.wikipedia.org/wiki/Heat_shockhttp://en.wikipedia.org/wiki/Nucleofectionhttp://en.wikipedia.org/wiki/Transduction_%28genetics%29http://en.wikipedia.org/wiki/Virushttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Plasmidhttp://en.wikipedia.org/wiki/Nanowireshttp://en.wikipedia.org/wiki/Siliconhttp://en.wikipedia.org/wiki/Carbon_nanofibershttp://en.wikipedia.org/wiki/Impalefectionhttp://en.wikipedia.org/wiki/Magnet_assisted_transfectionhttp://en.wikipedia.org/wiki/Magnetofectionhttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Inerthttp://en.wikipedia.org/wiki/Nanoparticlehttp://en.wikipedia.org/wiki/Gene_gun


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RNA transfection

RNA can also be transfected into cells to transiently express its coded protein, or to study RNAdecay kinetics. The latter application is referred as siRNA transfection orRNA silencing, andhas become a major application in research (to replace the "knock-down" experiments, to study

the expression of proteins, i.e. of Endothelin-1 with potential applications in gene-therapy.

A limitation of the silencing approach rely on the toxicity of the transfection for cells, and itssuspected effect on the expression of other genes/proteins.

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MicroinjectionMicroinjection refers to the process of using a glass micropipette to insert substances at

a microscopic or borderline macroscopic level into a single living cell. It is a simple mechanical process in

which a needle roughly 0.5 to 5 micrometers in diameter penetrates the cell membrane and/or

the nuclear envelope. The desired contents are then injected into the desired sub-cellular compartment

and the needle is removed. Microinjection is normally performed under a specialized optical

microscope setup called a micromanipulator. The process is frequently used as a vector in genetic

engineering and transgenics to insert genetic material into a single cell. Microinjection can also be used

in the cloning of organisms, and in the study of cell biology and viruses.
http://en.wikipedia.org/wiki/MRNA#Degradationhttp://en.wikipedia.org/wiki/MRNA#Degradationhttp://en.wikipedia.org/wiki/SiRNAhttp://en.wikipedia.org/wiki/Gene_knockdownhttp://en.wikipedia.org/wiki/Micropipettehttp://en.wikipedia.org/wiki/Microscopichttp://en.wikipedia.org/wiki/Macroscopichttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Cell_membranehttp://en.wikipedia.org/wiki/Nuclear_envelopehttp://en.wikipedia.org/wiki/Optical_microscopehttp://en.wikipedia.org/wiki/Optical_microscopehttp://en.wikipedia.org/wiki/Micromanipulatorhttp://en.wikipedia.org/wiki/Vector_(molecular_biology)http://en.wikipedia.org/wiki/Genetic_engineeringhttp://en.wikipedia.org/wiki/Genetic_engineeringhttp://en.wikipedia.org/wiki/Transgenicshttp://en.wikipedia.org/wiki/Genetic_materialhttp://en.wikipedia.org/wiki/Cloninghttp://en.wikipedia.org/wiki/Cloninghttp://en.wikipedia.org/wiki/Genetic_materialhttp://en.wikipedia.org/wiki/Transgenicshttp://en.wikipedia.org/wiki/Genetic_engineeringhttp://en.wikipedia.org/wiki/Genetic_engineeringhttp://en.wikipedia.org/wiki/Vector_(molecular_biology)http://en.wikipedia.org/wiki/Micromanipulatorhttp://en.wikipedia.org/wiki/Optical_microscopehttp://en.wikipedia.org/wiki/Optical_microscopehttp://en.wikipedia.org/wiki/Nuclear_envelopehttp://en.wikipedia.org/wiki/Cell_membranehttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Macroscopichttp://en.wikipedia.org/wiki/Microscopichttp://en.wikipedia.org/wiki/Micropipettehttp://en.wikipedia.org/wiki/Gene_knockdownhttp://en.wikipedia.org/wiki/SiRNAhttp://en.wikipedia.org/wiki/MRNA#Degradationhttp://en.wikipedia.org/wiki/MRNA#Degradation


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Subtypes

Pronuclear Injection

Pronuclear injection is a technique used to create transgenic organisms by injecting genetic material

into nucleus of a fertilized oocyte. This technique is commonly used to study the roleofgenes using mouse models.

Pronuclear Injection in Mice

In order for pronuclear injection to be successful, the genetic material (typically linear DNA) must be

injected while the genetic material from the oocyte and sperm are separate (i.e., the pronuclear phase).

In order to obtain these oocytes, mice are

commonly superovulated using gonadotrophins.]Once plugging has occurred, oocytes are harvested

from the mouse and injected with the genetic material. The oocyte is then implanted in the oviduct of

a pseudopregnant animal. While efficiency varies, 10-40% of mice born from these implanted oocytesmay contain the injected construct. Transgenic mice can then be bred to create transgenic lines.

Problems with Pronuclear Injection

The incorporation of injected DNA into the mouse genome cannot be tightly controlled. As a result,

improper incorporation may occur. If DNA is incorporated at the 2-cell stage, some cells may express the

DNA while others will not (so called mosaic mice). In addition, if the injected DNA is incorporated in

multiple sites, multiple copies of the DNA may be expressed leading to over expression. Even if only one
http://en.wikipedia.org/wiki/Transgenichttp://en.wikipedia.org/wiki/Organismhttp://en.wikipedia.org/wiki/Injection_(medicine)http://en.wikipedia.org/wiki/Geneticshttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Fertilizedhttp://en.wikipedia.org/wiki/Oocytehttp://en.wikipedia.org/wiki/Geneshttp://en.wikipedia.org/wiki/Mousehttp://en.wikipedia.org/wiki/Animal_modelhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Pronucleushttp://en.wikipedia.org/wiki/Ovarian_hyperstimulationhttp://en.wikipedia.org/wiki/Gonadotrophinshttp://en.wikipedia.org/wiki/Microinjection#cite_note-ucsandiego-2http://en.wikipedia.org/wiki/Microinjection#cite_note-ucsandiego-2http://en.wikipedia.org/wiki/Microinjection#cite_note-ucsandiego-2http://en.wikipedia.org/wiki/Mating_plughttp://en.wikipedia.org/wiki/Oviducthttp://en.wikipedia.org/wiki/Pseudopregnancyhttp://en.wikipedia.org/wiki/Animalhttp://en.wikipedia.org/wiki/DNA_constructhttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Gene_expressionhttp://en.wikipedia.org/wiki/Gene_expressionhttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/DNA_constructhttp://en.wikipedia.org/wiki/Animalhttp://en.wikipedia.org/wiki/Pseudopregnancyhttp://en.wikipedia.org/wiki/Oviducthttp://en.wikipedia.org/wiki/Mating_plughttp://en.wikipedia.org/wiki/Microinjection#cite_note-ucsandiego-2http://en.wikipedia.org/wiki/Gonadotrophinshttp://en.wikipedia.org/wiki/Ovarian_hyperstimulationhttp://en.wikipedia.org/wiki/Pronucleushttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Animal_modelhttp://en.wikipedia.org/wiki/Mousehttp://en.wikipedia.org/wiki/Geneshttp://en.wikipedia.org/wiki/Oocytehttp://en.wikipedia.org/wiki/Fertilizedhttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Geneticshttp://en.wikipedia.org/wiki/Injection_(medicine)http://en.wikipedia.org/wiki/Organismhttp://en.wikipedia.org/wiki/Transgenic


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copy of DNA is incorporated into the genome, if it is not incorporated into the germline, then it will not

be passed to offspring.

Protoplast Injection

Microcapillary and microscopic devices can be used to deliver DNA into a protoplast.
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Electroporation

Diagram of the major components of an electroporator with cuvette loaded.

Dr.Eberhard Neumann - Electroporation Founder

Electroporation, or electropermeabilization, is a significant increase in the electricalconductivity and permeability of the cell plasma membrane caused by an externally appliedelectrical field. It is usually used in molecular biology as a way of introducing some substanceinto a cell, such as loading it with a molecular probe, a drug that can change the cell's function,or a piece of coding DNA.

Electroporation is a dynamic phenomenon that depends on the local transmembrane voltage ateach point on the cell membrane. It is generally accepted that for a given pulse duration and

shape, a specific transmembrane voltage threshold exists for the manifestation of theelectroporation phenomenon (from 0.5 V to 1 V). This leads to the definition of an electric fieldmagnitude threshold for electroporation (Eth). That is, only the cells within areas where EEthare electroporated. If a second threshold (E ir) is reached or surpassed, electroporation willcompromise the viability of the cells, i.e., irreversible electroporation.

In molecular biology, the process of electroporation is often used for the transformation ofbacteria, yeast, and plant protoplasts. In addition to the lipid membranes, bacteria also havecell walls which are different from the lipid membranes and are made ofpeptidoglycan and itsderivatives. However, the walls are naturally porous and only act as stiff shells that protect

bacteria from severe environmental impacts. If bacteria and plasmids are mixed together, theplasmids can be transferred into the cell after electroporation. Several hundred volts across adistance of several millimeters are typically used in this process. Afterwards, the cells have tobe handled carefully until they have had a chance to divide producing new cells that containreproduced plasmids. This process is approximately ten times as effective as chemicaltransformation.
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This procedure is also highly efficient for the introduction of foreign genes in tissue culturecells, especially mammalian cells. For example, it is used in the process of producing knockoutmice, as well as in tumor treatment, gene therapy, and cell-based therapy. The process ofintroducing foreign DNAs into eukaryotic cells is known as transfection.

Electroporation is done with electroporators, appliances that create an electro-magnetic fieldin the cell solution. The cell suspension is pipetted into a glass or plastic cuvette which has twoaluminum electrodes on its sides.

For bacterial electroporation, typically a suspension of around 50 microliters is used. Prior toelectroporation it is mixed with the plasmid to be transformed. The mixture is pipetted into thecuvette, the voltage and capacitance are set, and the cuvette is inserted into theelectroporator. Immediately after electroporation, one milliliter of liquid medium is added tothe bacteria (in the cuvette or in an eppendorf tube), and the tube is incubated at the bacteria'soptimal temperature for an hour or more to allow recovery of the cells and expression ofantibiotic resistance, followed by spreading on agar plates.

The success of the elecroporation depends greatly on the purity of the plasmid solution,especially on its salt content. Solutions with high salt concentrations might cause an electricaldischarge (known as arcing), which often reduces the viability of the bacteria.

For a further detailed investigation of the process more attention should be paid to the outputimpedance of the porator device and the input impedance of the cells suspension (e.g. saltcontent). As the process needs direct electrical contact between the electrodes and thesuspension, and is inoperable with isolated electrodes, obviously the process involves certainelectrolytic effects, due to small currents and not only fields.

Electroporators

Electroporators come in two types: hand-held and bench-tops. Benchtop electroporators aregenerally used as common lab equipment, residing atop a central bench or hood. Some unitsoffer the possibility of electroporating multiple samples at the same time with special electrodeassemblies that fit into multi-well cell culture dishes. Benchtop electroporators can also be setto different operating parameters depending on whether or not the cell has a cell wall. Incontrast, there is a handheld electroporator available ("The Cloning Gun", a device created andmanufactured by Tritech Research). Separate units to electroporate either bacterial and

mammalian cells are cordless, rechargeable and use disposable pipectrodes, which combineelements of both cuvettes and pipettes. Their operating parameters are preset to the optimalparameters for transforming either bacteria or mammalian cells.

Both types of electoporators have been used on a wide range of cells - including E. coli (fortransformation) and mammalian cells such as neurons, astrocytes, neuroglia, lymphocytes,monocytes, fibroblasts, epithelial and endothelial cells from humans, mice, rats and monkeys(for transfection).
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Medical applications

A higher voltage of electroporation was found in pigs to irreversibly destroy target cells within anarrow range while leaving neighboring cells unaffected, and thus represents a promising newtreatment for cancer, heart disease and other disease states that require removal of tissue.

A recent technique called non-thermal irreversible electroporation (N-TIRE) has provensuccessful in treating many different types of tumors and other unwanted tissue. Thisprocedure is done using small electrodes (about 1mm in diameter), placed either inside orsurrounding the target tissue to apply short, repetitive bursts of electricity at a predeterminedvoltage and frequency.

These bursts of electricity increase the resting transmembrane potential (TMP), so thatnanopores form in the plasma membrane. When the electricity applied to the tissue is abovethe electric field threshold of the target tissue, the cells become permanently permeable from

the formation of nanopores. As a result, the cells are unable to repair the damage and die dueto a loss of homeostasis. N-TIRE is unique to other tumor ablation techniques in that it does notcreate thermal damage to the tissue around it.

Contrastingly, reversible electroporation occurs when the electricity applied with the electrodesis below the electric field threshold of the target tissue. Because the electricity applied is belowthe cells threshold, it allows the cells to repair their phospholipid bilayer and continue on withtheir normal cell functions. Reversible electroporation is typically done with treatments thatinvolve getting a drug or gene (or other molecule that is not normally permeable to the cellmembrane) into the cell. Not all tissue has the same electric field threshold; therefore carefulcalculations need to be made prior to a treatment to ensure safety and efficacy.

One major advantage of using N-TIRE is that, when done correctly according to carefulcalculations, it only affects the target tissue. Proteins, the extracellular matrix, and criticalstructures such as blood vessels and nerves are all unaffected and left healthy by thistreatment. This allows for a quicker recovery, and facilitates a more rapid replacement of deadtumor cells with healthy cells.

The first successful treatment of malignant cutaneous tumors implanted in mice wascompleted in 2007 by a group of scientists who achieved complete tumor ablation in 12 out of13 mice. They accomplished this by to sending 80 pulses of 100 microseconds at 0.3Hz with an

electrical field magnitude of 2500 V/cm to treat the cutaneous tumors.

Before doing the procedure, scientists must carefully calculate exactly what needs to be done,and treat each patient on an individual case-by-case basis. To do this, imaging technology suchas CT scans and MRIs are commonly used to create a 3D image of the tumor. From this

information, they can approximate the volume of the tumor and decide on the best course ofaction including the insertion site of electrodes, the angle they are inserted in, the voltageneeded, and more, using software technology. Often, a CT machine will be used to help with
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the placement of electrodes during the procedure, particularly when the electrodes are beingused to treat tumors in the brain.

The entire procedure is very quick, typically taking about five minutes. The success rate of theseprocedures is high and is very promising for future treatment in humans. One disadvantage to

using N-TIRE is that the electricity delivered from the electrodes can stimulate muscle cells tocontract, which could have lethal consequences depending on the situation. Therefore, aparalytic agent must be used when performing the procedure. The paralytic agents that havebeen used in such research are successful; however, there is always some risk, albeit slight,when using anesthetics.

A more recent technique has been developed called high-frequency irreversible electroporation(H-FIRE). This technique uses electrodes to apply bipolar bursts of electricity at a highfrequency, as opposed to unipolar bursts of electricity at a low frequency. This type ofprocedure has the same tumor ablation success as N-TIRE. However, it has one distinctadvantage, H-FIRE does not cause muscle contraction in the patient and therefore there is noneed for a paralytic agent.

Scientists have had high success rates in treating lab animals with many different kinds oftumors. Although scientists are not ready to routinely use irreversible electroporation to treatcomplicated tumors in humans, N-TIRE is more commonly used in humans to treat simplecutaneous or subcutaneous tumors.

In addition to treating cutaneous and subcutaneous tumors, there have been attempts at usingthis technology to treat prostate, lung, kidney and liver cancer in humans. However, scientistsare still in the process of understanding this technology and its affect on animals and humans.

One study done by Kenneth Thompson et al.[

tested the safety of N-TIRE on humans sufferingfrom lung, kidney or liver tumors. Of the 69 tumors treated, 49 of them achieved completetumor ablation. The most successful treatment rate occurred in liver tumors. This studyprovides encouraging evidence for the future of the use of N-TIRE as a method of treatingcancer in humans.

Electroporation can also be used to help deliver drugs or genes into the cell by applying shortand intense electric pulses that transiently permeabilize cell membrane, thus allowing transportof molecules otherwise not transported through a cellular membrane. This procedure isreferred to as electrochemotherapy when the molecules to be transported is achemotherapeutic agent or gene electrotransfer when the molecule to be transported is DNA.

Physical mechanism

Electroporation allows cellular introduction of large highly charged molecules such as DNAwhich would never passively diffuse across the hydrophobic bilayer core. This phenomenonindicates that the mechanism is the creation of nm-scale water-filled holes in the membrane.Although electroporation and dielectric breakdown both result from application of an electric
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field, the mechanisms involved are fundamentally different. In dielectric breakdown the barriermaterial is ionized, creating a conductive pathway. The material alteration is thus chemical innature. In contrast, during electroporation the lipid molecules are not chemically altered butsimply shift position, opening up a pore which acts as the conductive pathway through thebilayer as it is filled with water.

Electroporation is a multi-step process with several distinct phases. First, a short electrical pulsemust be applied. Typical parameters would be 300-400 mV for < 1 ms across the membrane(note- the voltages used in cell experiments are typically much larger because they are beingapplied across large distances to the bulk solution so the resulting field across the actualmembrane is only a small fraction of the applied bias).

Upon application of this potential the membrane charges like a capacitor through the migrationof ions from the surrounding solution. Once the critical field is achieved there is a rapidlocalized rearrangement in lipid morphology. The resulting structure is believed to be a pre -pore since it is not electrically conductive but leads rapidly to the creation of a conductivepore.

Evidence for the existence of such pre-pores comes mostly from the flickering of pores, whichsuggests a transition between conductive and insulating states. It has been suggested thatthese pre-pores are small (~3 ) hydrophobic defects. If this theory is correct, then thetransition to a conductive state could be explained by a rearrangement at the pore edge, inwhich the lipid heads fold over to create a hydrophilic interface.

Finally, these conductive pores can either heal, resealing the bilayer or expand, eventuallyrupturing it. The resultant fate depends on whether the critical defect size was exceeded which

in turn depends on the applied field, local mechanical stress and bilayer edge energy.
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Gene gun

A gene gun or a biolistic particle delivery system, originally designed forplant transformation,is a device for injecting cells with genetic information. The payload is an elementalparticle of a

heavy metal coated with plasmid DNA. This technique is often simply referred to asbioballistics orbiolistics.

This device is able to transform almost any type of cell, including plants, and is not limited togenetic material of the nucleus: it can also transform organelles, including plastids.

Design

The gene gun was originally a Crosman air pistol modified to fire dense tungstenparticles. It wasinvented by John C Sanford, Ed Wolf and Nelson Allen at Cornell University, and Ted Klein ofDuPont, between 1983 and 1986. The original target was onions (chosen for their large cell size)

and it was used to deliver particles coated with a marker gene. Genetic transformation was thenproven when the onion tissue expressed the gene.

The earliest custom manufactured gene guns (fabricated by Nelson Allen) used a 22 calibernailgun cartridge to propel an extruded polyethylene cylinder (bullet) down a 22 cal. Douglas barrel.A droplet of the tungsten powder and genetic material was placed on the bullet and shot downthe barrel at a lexan "stopping" disk with a petri dish below. The bullet welded to the disk andthe genetic information blasted into the sample in the dish with a doughnut effect (devastation inthe middle, a ring of good transformation and little around the edge). The gun was connected to avacuum pump and was under vacuum while firing.

The early design was put into limited production by a Rumsey-Loomis (a local machine shopthen at Mecklenburg Rd in Ithaca, NY, USA). Later the design was refined by removing the"surge tank" and changing to nonexplosive propellants. DuPont added a plastic extrusion to theexterior to visually improve the machine for mass production to the scientific community. Bioradcontracted with Dupont to manufacture and distribute the device. Improvements include the useofheliumpropellant and a multi-disk-collision delivery mechanism. Other heavy metals such asgold and silverare also used. Gold may be favored because it has better uniformity than tungstenand tungsten can be toxic to cells, but its use may be limited due to availability and cost.

Application

Gene guns are so far mostly applied for plant cells. However, there is much potential use inanimals and humans as well.

Plants

The target of a gene gun is often a callus of undifferentiated plant cells growing on gel mediumin a petri dish. After the gold particles have impacted the dish, the gel and callus are largelydisrupted. However, some cells were not obliterated in the impact, and have successfully
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enveloped a DNA coated gold particle, whose DNA eventually migrates to and integrates into aplant chromosome.

Cells from the entire petri dish can be re-collected and selected for successful integration andexpression of new DNA using modern biochemical techniques, such as a using a tandem

selectable gene and northern blots.

Selected single cells from the callus can be treated with a series of plant hormones, such asauxins 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 newplant that originated from a successfully shot cell may have new genetic (heritable) traits.

The use of the gene gun may be contrasted with the use ofAgrobacterium tumefaciensand its Tiplasmid to insert genetic information into plant cells. See transformation for different methods oftransformation in different species.

Humans and animals

Gene guns have also been used to deliverDNA vaccines.

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

The gene gun technique is also popularly used in an edible vaccine production technique, wherethe nano-gold particles coated with plant genetic material under the high vacuum pressurizedchamber are transformed into suitable plant tissues.

The gene gun has become a common tool for labeling subsets of cells in cultured tissue. Inaddition to being able to transfect cells with DNA plasmids coding for fluorescent proteins, thegene gun can be adapted to deliver a wide variety of vital dyes to cells.

Gene gun bombardment has also been used to transform C. elegans, as an alternative tomicroinjection.
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Somatic fusion

Somatic fusion, also called protoplast fusion, is a type of genetic modification in plants bywhich two distinct species of plants are fused together to form a new hybrid plant with the

characteristics of both, a somatic hybrid. Hybrids have been produced either between thedifferent varieties of the same species (e.g. between non-flowering potatoplants and floweringpotato plants) or between two different species (e.g. between wheat triticum and rye secale toproduce Triticale).

Uses of somatic fusion include making potato plants resistant to potato leaf roll disease. Throughsomatic fusion, the crop potato plant Solanum tuberosumthe yield of which is severely reducedby a viral disease transmitted on by the aphid vectoris fused with the wild, non-tuber-bearingpotato Solanum brevidens, which is resistant to the disease. The resulting hybrid has thechromosomes of both plants and is thus similar to polyploidplants. Somatic Hybridization wasfirstly introduced by CarlsoninNicotiana 'glauea'

Process for plant cells

The somatic fusion process occurs in four steps:

The removal of the cell wall of one cell of each type of plant using cellulase enzyme to producea somatic cell called a protoplast

1. The cells are then fused using electric shock (electrofusion) or chemical treatment to join thecells and fuse together the nuclei. The resulting fused nucleus is called heterokaryon.

2. The somatic hybrid cell then has its cell wall induced to form using hormones3. The cells are then grown into calluses which then are further grown to plantlets and finally to afull plant, known as a somatic hybrid.

Different from the procedure for seed plants describe above, fusion of mossprotoplasts can beinitiated without electric shock but by the use ofpolyethylene glycol (PEG). Further, mossprotoplasts do not need phytohormones forregeneration, and they do not form a callus. Instead,regenerating moss protoplasts behave like germinating moss spores.[4] Of further note sodiumnitrate and calcium ion at high pH can be used, although results are variable depending on theorganism.

Applications in animal cells

Somatic cells of different types can be fused to obtain hybrid cells. Hybrid cells are useful in avariety of ways, e.g.,

(i) To study the control ofcell division and gene expression,

(ii) To investigate malignant transformations,
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(iii) to obtain viral replication,

(iv) forgene orchromosome mapping and for

(v) production of monoclonal antibodies by producing hybridoma (hybrid cells between an

immortalised cell and an antibodyproducing lymphocyte), etc.

Chromosome mapping through somatic cell hybridization is essentially based on fusion ofhuman and mouse somatic cells. Generally, human fibrocytes orleucocytes are fused with mousecontinuous cell lines.

When human and mouse cells (or cells of any two mammalian species or of the same species)are mixed, spontaneous cell fusion occurs at a very low rate (10-6). Cell fusion is enhanced 100to 1000 times by the addition of ultraviolet inactivated Sendai (parainfluenza) virus orpolyethylene glycol (PEG).

These agents adhere to the plasma membranes of cells and alter their properties in such a waythat facilitates their fusion. Fusion of two cells produces a heterokaryon, i.e., a single hybrid cellwith two nuclei, one from each of the cells entering fusion. Subsequently, the two nuclei alsofuse to yield a hybrid cell with a single nucleus.

A generalized scheme for somatic cell hybridization may be described as follows. Appropriatehuman and mouse cells are selected and mixed together in the presence of inactivated Sendaivirus or PEG to promote cell fusion. After a period of time, the cells (a mixture of man, mouseand 'hybrid' cells) are plated on a selective medium, e.g., HAT medium, which allows themultiplication of hybrid cells only.

Several clones (each derived from a single hybrid cell) of the hybrid cells are thus isolated andsubjected to both cytogenetic and appropriate biochemical analyses for the detection ofenzyme/protein/trait under investigation. An attempt is now made to correlate the presence and absenceof the trait with the presence and absence of a human chromosome in the hybrid clones.

If there is a perfect correlationbetween the presence and absence of a human chromosome andthat of a trait in the hybrid clones, the gene governing the trait is taken to be located in theconcerned chromosome.

The HAT medium is one of the several selective media used for the selection of hybrid cells.This medium is supplemented with hypoxanthine, aminopterin and thymidine, hence the name

HAT medium. Antimetabolite aminopterin blocks the cellular biosynthesis ofpurines andpyrimidines from simple sugars and amino acids.

However, normal human and mouse cells can still multiply as they can utilize hypoxanthine andthymidine present in the medium through a salvage pathway, which ordinarily recycles thepurines and pyrimidines produced from degradation ofnucleic acids.
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Hypoxanthine is converted into guanine by the enzyme hypoxanthine-guaninephosphoribosyltransferase (HGPRT), while thymidine is phosphorylated by thymidine kinase(TK); both HGPRT and TK are enzymes of the salvage pathway.

On a HAT medium, only those cells that have active HGPRT (HGPRT+) and TK (TK+)

enzymes can proliferate, while those deficient in these enzymes (HGPRr- and/or TK-) can notdivide (since they cannot produce purines and pyrimidines due to the aminopterin present in theHAT medium).

For using HAT medium as a selective agent, human cells used for fusion must be deficient foreither the enzyme HGPRT or TK, while mouse cells must be deficient for the other enzyme ofthis pair. Thus, one may fuse HGPRT deficient human cells (designated as TK+ HGPRr-) withTK deficient mouse cells (denoted as TK- HGPRT+).

Their fusion products (hybrid cells) will be TK+ (due to the human gene) and HGPRT+ (due tothe mouse gene) and will multiply on the HAT medium, while the man and mouse cells will fail

to do so. Experiments with other selective media can be planned in a similar fashion.

Characteristics of Somatic Hybridization and Cybridization

1. Somatic cell fusion appears to be the only means through which two different parentalgenomes can be recombined among plants that cannot reproduce sexually (asexual or sterile).

2. Protoplasts of sexually sterile (haploid, triploid, and aneuploid) plants can be fused to producefertile diploids and polyploids.

3. Somatic cell fusion overcomes sexual incompatibility barriers. In some cases somatic hybrids

between two incompatible plants have also found application in industry oragriculture.

4. Somatic cell fusion is useful in the study of cytoplasmic genes and their activities and thisinformation can be applied in plant breeding experiments.
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Ti plasmid

The structure of the Ti plasmid

A Ti ortumour inducing plasmid is a circular plasmid that often, but not always, is a part of thegenetic equipment that Agrobacterium tumefaciens and Agrobacterium rhizogenes use totransduce its genetic material to plants. The Ti plasmid is lost when Agrobacterium is grownabove 28C. Such cured bacteria do not induce crown galls, i.e. they become avirulent. pTi andpRi share little sequence homology but are functionally rather similar. The Ti plasmids areclassified into different types based on the type of opineproduced by their genes. The differentopines specified by pTi are octopine, nopaline, succinamopine and leucinopine.

The plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. Theplasmid is 206,479 nucleotides long, the GC content is 56% and 81% of the material is codinggenes. There are no pseudogenes.

The modification of this plasmid is very important in the creation oftransgenic plants.

Virulence Region

Genes in the virulence region are grouped into the operons virABCDEFG, which code for theenzymes responsible for mediating transduction of T-DNA to plant cells.

virA codes for a receptor which reacts to the presence of phenolic compounds such asacetosyringone, syringealdehyde oracetovanillone which leak out of damaged plant tissues.

virB encodes proteins which produce a pore/pilus-like structure. virCbinds the overdrive sequence.[ virD1 and virD2 produce endonucleases which target the direct repeat borders of the T-DNA

segment,
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vir E Binds to T-strand protecting it from nuclease attack, and intercalates with lipids to formchannels in the plant membranes through which the T-complex passes, beginning with the rightborder.

virG (TRANSCRIPTIONAL FACTOR) activates vir-gene expression after binding to a consensussequence, once it has been phosphorylated by virA.[

Characteristics Features

Agrobacterium is called the natural genetic en

M.sc.Micro 3rd Sem Unit 3

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