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PTC - C11 (Genetic Transformation) Cheat Sheet (DRAFT) by

Brief summary of Chapter 11 (Genetic Transformation) of Plant and Tissue Culture Subject

This is a draft cheat sheet. It is a work in progress and is not finished yet.

Introd­uction

Genetic transf­orm­ation is a process that involves the introd­uction and expression of foreign genes in a host organism
This expression can result from the extrac­hro­mos­omal, or episomal, presence of genes in nuclei that may persist if the introduced DNA has a mechanism for replic­ation

Genetic Transf­orm­ation Methods

1. Using Calcium Phosphate
2. Microi­nje­ction
3. Lipofe­ction
4. Electr­opo­ration
5. Bombar­dment
6. Polyet­hylene glycol (PEG)-­med­iated transf­orm­ation
7. Agroba­cterium mediated transf­orm­ation

1. Calcium Phosphate

-HEPES­-bu­ffered saline solution is mixed with a calcium chloride solution containing DNA for transf­ection to form a fine precip­itate of calcium phosphate with DNA.
-The suspension of the precip­itate is then added to the monolayer of cells.
-The cells take up the calciu­m-p­hos­pha­te-DNA complexes by endocy­tosis and express genes.

Calcium Phosphate Transf­orm­ation

2. Microi­nje­ction

-DNA is directly injected into the nucleus using a fine glass capillary under a micros­cope.
-However this method acquire a great effort as each and every cell has to be injected indivi­dually.

Microi­nje­ction Process

3. Lipofe­ction

-use of cationic lipids for DNA transf­ection into mammalian cells
-safer than viral vectors
-can be produced in large quantities
-can deliver large DNA fragments of up to several megabase pairs long into cells
-There are many formul­ations of lipid reagents for transf­ection, but they normally contain a positively charged moiety attached to a neutral lipid component.
a) On mixing of these reagents with DNA, the charged head groups are drawn towards the phosphate backbone of DNA and form lipid-DNA complexes.
b) When the suspension of these complexes is added to the cells, the positively charged head groups of the lipid are attracted to the negatively charged cell membrane.
c) The end-result is that the lipid-DNA complex is either fused to the cell membrane or enters the cell by endocy­tosis, transf­erring its DNA load into the cell.

Lipofe­ction Process

4. Electr­opo­ration

-Host cells and selected molecules are suspended in a conductive solution, and an electrical circuit is closed around the mixture.
-An electrical pulse at an optimized voltage and only lasting a few micros­econds to a millis­econd is discharged through the cell suspen­sion.
-This disturbs the phosph­olipid bilayer of the membrane and results in the formation of temporary pores.
-The electric potential across the cell membrane simult­ane­ously rises to allow charged molecules like DNA to be driven across the membrane through the pores
-This technique can used for protop­last, intact cell & tissue (callus culture, immature embryos, influo­res­cence material)
-Effic­iency is depend on condition of plant and tissue treatment conditions chosen.
-Linear DNA may can improve efficiency of electr­opo­ration
 
Process
1. Material incubated in a buffer solution containing DNA and subjected to contro­lled, millis­econd high-v­oltage electrical pulses 100-200 V for 1-2 ms
2. High-v­oltage- induce transient pore in the cell membrane and allow DNA migrate through plasma membrane and integrate with genome.
3. After pulsing, cell membrane reseals and left unharmed.
4. Plant materials may require pre- and post-e­lec­tro­por­ation incubation in buffer of high osmotic pressure.
 
Advantages
Disadv­antages
1. Produced transf­ormants with low transgene copy numbers
1. Low effici­ency; requires careful optimi­zation
2. High deliver rate
-
3. Transf­ormed cells will not damage due to transf­orm­ation
-
*Proto­plasts are cells stripped of their cell walls and maintained in culture
*Transgene copy numbers is defined as the number of exogenous DNA insert(s) in the genome.

Electr­opo­ration Process

5. Bombar­dment

Principle: Using a gene gun directly shoot a piece of DNA into recipient plant tissue.
Also known as: Biolis­tics, Particle bombar­dment, Microp­roj­ectile bombar­dment, Particle inflow gun
-Particles should be high enough mass in order to possess adequate momentum to penetrate into plant cell and achieve particle delivery to plant cells
-Metals should be chemically inert to prevent adverse reaction with DNA and cell component. Eg. gold, tungsten, palladium, rhodium, platinum and iridium
-Plant cell are competent cell for transf­orm­ation
-After bombar­dment, cells require a “healing” period under special condition of light, temper­ature, and humidity.
 
Process
1. Separation of the protoplast from leaf
2. DNA-coated microc­arriers are loaded on the macroc­arrier
3. Microc­arriers are shot towards target tissue during helium gas decomp­res­sion.
4. A stopping screen placed allowing the DNA-coated microc­arriers to pass through and reach the target.
5. Transfer to the solid media
6. Transfer of the transgenic plant in a greenhouse
 
Advantage
1. Unlimited host range
2. Not limited by ability to regenerate from single cells
3. Immature embryos from seeds will continue to develop
4. Transgenic plants selected
 

Bombar­dment Process

6. PEG-Me­diated Transf­orm­ation

PEG: Polyet­hylene glycol
-Trans­for­mation of naked DNA done by treatment with PEG in presence of divalent cations
-PEG and divalent cations destab­ilize the plasma membrane of plant protoplast and render it permeable to naked DNA.
 
Advantage
Disadv­antage
1. simple and efficient, allowing a simult­aneous processing of many samples
1. Plant protop­lasts are not easy to work with, and the regene­ration of fertile plants from protop­lasts is proble­matic for some species.
2. yields a transf­ormed cell population with high survival and division rates
2. The DNA used is also suscep­tible to degrad­ation and rearra­nge­ment.
3. helps to overcome a hurdle of host range limita­tions of Agroba­cte­riu­m-m­ediated transf­orm­ation.

7. Agroba­cte­riu­m-m­ediated Transf­orm­ation

-ability of an organism to transfer its T-DNA into the host cells effici­ently
-compo­nents: T-DNA present on the plasmid called Ti (tumor­-in­ducing) plasmid along with other functional components like virulence (vir), conjug­ation (con), and origin of rep­lic­ati­on ­(ori); T-DNA consists of 25 bp repeats that end at the T-region & virulence (vir) region composed of seven major loci
-transfer of a piece of plasmid by the bacteria into the plant cells during infection
-plasmid then integrates into the nuclear genome in order to express its own genes and affect the hormonal balance in the host cell
-bacteria also produce a number of enzymes that are involved in the synthesis of opines that is then used by the bacteria as nutrients
 
Bacterial Infection Process:
1. entry of the bacteria through wounded sites
2. The binding of bacteria to the plant cells is enhanced by the release of phenolic acetos­yri­ngone (AS) by the injured plant cells
3. The AS activates the VirA proteins on the bacteria, which activates VirG via phosph­ory­lation of its aspartate residue.
4. The activated form of VirG then binds to other vir genes, inducing their expres­sion. VirD activated by this process stimulates the T-strand generation (a single­-st­randed copy of the T-DNA).
5. The VirD2 covalently binds to the 5’ end of the T-strand as the 5’ end is the leading end during the transfer. Other factors like VirE2 and VirB proteins also bind to the T-strand, forming a T-complex.
6. The complex is then passed into the nucleus by the nuclear target signals released by the Vir proteins.
7. T-DNA strand is integrated into the plant genome randomly as either a single copy or multiple copies
8. The integr­ation usually occurs in the transc­ription active or repetitive regions of the genome by the process of recomb­ina­tion.

Agroba­cte­riu­m-m­ediated transf­orm­ation

Plasmid Cloning Vectors

Agroba­cte­riu­m-m­ediated Transf­orm­ation of Tobacco

5 basic protocols used for any Agroba­cte­riu­m-m­ediated transf­orm­ation in tobacco
1. Suitable tobacco plant tissue
-in this case leaves must be removed from a donor plant and sterilized to be used as explants source.
2. Co-cul­tiv­ation
-Cutting the leaf tissue into smaller pieces, placing it into culture of Agroba­cterium for approx­imately 30 minutes.
 
-During this incubation period, the bacteria will attach to the plant cells.
 
-Remove the explants and blot the excess bacterial culture off and then place into solid Murashige and Skoog (MS) medium with no selective agent.
3. Incubate MS medium with the explants for 2 days - T-DNA can be transf­erred to plant cells.
-
4. Remove explants from the medium and wash in antibiotic solution to kill the Agroba­cterium cells.
-
5. Transfer explants to fresh solid medium with a few selective agents
-(kana­mycin) So that growth of non-tr­ans­formed plant cells can be inhibited
 
-(cefo­taxime) So that growth of any extra surviving Agroba­cterium can be killed.
 
-Auxins and cytokinins added.

Genetic Transf­orm­ation Screening

1. Blue white screening
-DNA of interest is ligated into a vector. The vector is then transf­ormed into competent bacterial cells. The competent cells are grown in the presence of X-gal. If the ligation was succes­sful, the bacterial colony will be white; if not, the colony will be blue. This technique allows for the quick and easy detection of successful ligation
2. Restri­ction enzyme screening
-First, restri­ction mapping should be performed to identify which restri­ction enzymes can be used to easily identify the presence of your insert within the plasmid. After isolating a plasmid DNA from an overnight bacterial culture, digest the purified plasmid DNA from recomb­inant clones using restri­ction enzymes. Once digested, run the plasmid on an agarose gel to verify that the vector backbone and insert are of the expected sizes
3. Antibiotic resistance screening
-After transf­orm­ation, cells are grown in a medium containing the said antibi­otics to screen out transf­ormants carrying antibiotic resistance gene and gene of interest.
 
Reasons for screening after gene transf­orm­ation
1. To identify transf­ormants with the gene insert of interest from those without gene insert of interest in the vector transf­ormed into the host
2. To identify for sense and antisense gene insert in the vector transf­ormed into the host
3. To identify host that expresses the gene of interest from those that does not expresses the gene of interest