r DNA Tchnology

8/6/2019 r DNA Tchnology

1/32

RECOMINANT DNA TECHNOLOGY

GENETIC ENGINEERING (manipulation) provides technique for investigation and

alteration of gene structure and function. The alteration in gene structure is done with aim to

produce the desired characteristics and functions. Molecular biology has lead directly to

immensely powerful and potentially profitable techniques for genetic engineering. A great dealof effort is being directed into genetic engineering or manipulation of microorganisms to make

them produce a range of valuable polypeptides such as insulin, blood clotting factor VIII, growth

hormones which they would not normally produce and which are expensive to prepare by

conventional biochemical methods. It is easy and cheap to grow microorganism on the large

scale. There are very attractive as potential sources of these polypeptides. Genetic manipulation

of plants and livestock should also permit the introduction of beneficial characteristics such as

resistance to herbicides or diseases that could not be readily obtain by conventional breeding.

Theunderstanding of gene structure has made it possible to understand the ways in which call

store and express genetic information, which can be manipulated according to needs. Genemanipulation requires cuttings and joining lengths of DNA in a precise way. This is

alternately called recombinant DNA technology.

The DNA technology is a part of molecular genetics. It involves the cutting of DNA into

specific fragments and joining these fragments using various enzymes. During this process there

is transfer of genetic material from one organism to other organism.

Requirements: There are basic three requirements for this technology.

(1) Vehicle DNA- It is a DNA used to carry foreign DNA molecule and then transferring it

molecule into the most cell.

(2)Passenger DNA. It is a DNA molecule (foreign DNA) which is being transferred from

one organism to another organism with the help of vehicle DNA.

(3)Enzymes-There are biocatalysts required for cutting and joining of DNA molecule.

Steps principal involved in DNA technology

(1) DNA fragments coding for proteins of interest are synthesized either chemically or isolatedfrom an organism with the help of enzymes. These DNA fragment are called passenger

DNA.

(2) These DNA fragments (passenger DNAS) are inserted into vehicle DNAs with the help of

enzymes restriction endonucleases and ligases etc.

1 | P a g e


2/32

A DNA molecule obtained after combination of vehicle DNA and passenger DNA is

called recombinant DNAs (r DNAs).

(3) The r DNA molecules are than introduced into a host cell to replicate.

Generally three methods are than used to introduce into r DNA to host cell.

(a) Transformation (b) Transduction (c) Conjugation.

(a) Transformation- It is a process by which a cell takes a naked DNA segment

the environment, incorporated it into its own chromosome DNA.

(b) Transduction- It is transfer of genetic material or r DNA from one organism

to another with the help of bacteriophage.

(c) Conjugation- The transfer of genetic material from donor to recipient bydirect contact.

(4) In the recipient host cells, r DNA replicates indefinitely.

In this way genes are cloned to provide sufficient material for detailed analyses or for

insertion into genome of a cell i.e., to be genetically engineer.

The principal steps involved in gene cloning are shown below

TOOLS OF RECOMBINANT DNA TECHNOLOGY

Various tools like enzymes, vectors and host organisms are required for carrying on the

recombinant DNA technology.

A) Enzymes: Various types of enzymes required are:

i) Restriction enzymes: Restriction enzymes are popularly called Molecular Scissors.

These are mainly classified as restriction endonucleases and restriction exonucleases.

Restriction exonucleases: They remove nucleotides from the ends of DNA. These digest

base pairs from 5 to 3 ends and act on single stranded DNA only.

2 | P a g e


3/32

Restriction endonucleases: They act upon genetic material and cleave the double stranded

DNA at any point except the ends, but they act on only one strand of duplex.

All restriction endonucleases have specific recognition sequence within the DNA

molecule. The sequences generally fall between four to six base pairs in length. The

restriction recognition sequences are often known as Pallindromic sequences i.e., thetwo strands are identical when both in forward and backward direction, in this region

read. Each individual enzyme recognizes only one specific sequence. However, the same

specific sequence is recognized by two or more different enzymes. Such types of

enzymes are called isochizomers.

These enzymes are called Natures Pinking Shears (Saw tooth blades)because these

occur naturally in bacteria as a chemical weapon against invading viruses and cut both

strands of DNA when certain foreign nucleotides are introduced in the cell.

Types of restriction endonucleases

Restriction endonucleases can be divided into three types: Type I, Type II and Type III.

Type I: The type I enzymes recognizes specific sequence but cut the DNA molecule far

away from the recognition sequence. The cleavage takes place about 1000 base pairs

away from recognition sit. Hence, the use of class I enzyme in genetic engineering is

restricted.

E.g. Eco K, Eco B etc.

Type II: These are able to recognize specific sequence and cut the DNA molecule from

at the recognition site only. Hence, this class of enzymes is the major choice in genetic

engineering work. Because majority of type II restriction enzymes recognize tetra, penta

and hexa and sometimes hepta nucleotide sequence. They require Mg 2+ for restriction

digestion. More than 350 different type II endonucleases with over 100 different

recognition sequences are known. The classical example for class II type restriction

enzyme is EcoR I. The first type II endonuclease discovered in 1960 was Hind II.

Type III: These are intermediate between type I and type II enzymes. They cleave the

DNA near to the recognition site. E.g Eco P1, EcoP15 etc. They recognize asymmetric

target site and cleave the DNA duplex on one side of the recognition sequence up to 20

base pairs away.

ii) Ligases- Though cutting of DNA very useful for DNA analyses. Its full potential is revealed

only when fragments produced are joining together to give new of called recombinant DNA.

This joining or ligation is achieved by use of DNA ligases enzymes. Two different DNA

3 | P a g e


4/32

preparations are treated with some enzymes to give fragments with sticky ends. These, when two

fragments are mixed, base pairing between sticky ends will result in coming together of

fragments. All these pairings are temporary owing to weakness of H-bonding between few bases

in the sticky ends .But they can be stabilize by use of DNA ligase which forms covalent bond

between 5 Po43- gp at end of one strand and 3 OH group of adjacent strand. This reaction is

driven by ATP and carried out at 10oc to lower K.E of molecules. So, reducing the changes of

base pairs sticky ends separating before they have not stabilized by ligation. However long

reaction times are needed to compensate for low activity of DNA ligase in the cold.

SIGNIFICANCE OF RESTRICTION ENZYMES:

1. Restriction enzymes are used in biotechnology to cut DNA into smaller strands in order

to study fragment length differences among individuals (Restriction fragment length

polymorphism-RFLP) or for gene cloning.

2. RFLP techniques have been used to determine that individuals have distinctivedifferences in gene sequences and restriction cleavage patterns in certain areas of the

genome.

3. Knowledge of these unique areas is the basis for DNA fingerprinting. Each of these

methods depends on the use of agarose gel electrophoresis for the separation of the DNA

fragments.

Cloning vectors- In principle, DNA is introduced into a suitable host call using bacteria like

E.coli. Where it is replicated as cell grows and divides. However, replication will occur only if

DNA contains a sequence i.e., recognized by cell as origin of replication. Most DNA samples do

not contain such sequence therefore DNA has to be cloned has to attach to a carrier or vector

DNA that does contain an origin of replication. Hence, vector is a DNA molecule that has the

ability to replicate in an appropriate host cell and into which the DNA fragment (passenger

DNA) is inserted for cloning. It is also called vehicle DNA.

Properties of a good vector

1. It should be able to replicate.

2. The vector should be easy to isolate and purify.

3. It should be easily introduced into the host cells.

4. The vector should have suitable marker genes that allow easy detection or / and selection of

the transformed host cells.

5. A vector should contain unique target sites for restriction endonucleases.

4 | P a g e


5/32

6. It should have ability to accommodate foreign DNA of various sizes without damage to

replicate functions.

Number of vectors can be employed for this purpose;-

1) PLASMID-

Plasmids that are used this cloning have following properties;-

(1) They are small and size varies from 1Kb to over 25 KB.

(2) They are present in high copy number e.g., An E.coli may contain up to 7 different kinds of

plasmids.

(3) They contain suitable marker/ markers e.g, Antibiotic resistant marker

(4) They are not transmissible i.e. they are not transferred from one cell to another.

(5) They contain suitable restriction site that can be used for inserting DNA fragment for

cloning.

(6) They are stably maintained during replication in the host i.e. during cell division they are

segregated evenly in daughter cell.

(7) They can replicate in suitable hosts.

Not being a part of the main genome, the plasmids can be easily isolated and transferred to

another bacteria or organisms. These features make the plasmid suitable for use as vector or

vehicle DNA. A number of plasmids have been isolated from different hosts and several has

properties listed above and can be used as suitable cloning vector. These are simplest cloning

vectors and most widely used for gene cloning.A plasmid integrated to a chromosome is called

an Episome. One of most widely used plasmid is PBR-322.

These are called Natures interpolers. Because they are known to move around freely

in bacterial world and may pick up genes from one bacterium and transfer them to another,

thus providing variability to the asexually multiplying bacteria.

2) BACTERIOPHAGE

5 | P a g e


6/32

Bacteriophages are viruses that attack bacteria. Hence, are also called bacterial viruses.

Phage contains a proteinaceous head and a long tail attached to the head.

Several bacteriophages are used as cloning vectors, the most commonly used E.Coli

phages being (lambda) and M13 phages. Plasmid vectors have to be introduced into bacterial

cells, which are then cloned and selected for recovery of recombinant DNA. In contrast, thephage vectors are directly tested on an appropriatebacterial lawn (a continuous bacterial growth

on an agar plate) where each phage particle forms a plaque (a clear bacteria free zone in the

bacterial lawn).

Phage vectors present two advantages over plasmid vectors.

i) They are more efficient than plasmids for cloning of large DNA fragments; the largest

cloned insert size in a vector is just over 24 KB, while that for plasmid vectors it is

less than 15 Kb.

ii) It is easier to screen a large number of phage plaques than bacterial colonies for the

identification of recombinant vector.

Specific DNA sequences are present in vector are known as cos sites. These enable the DNA to

be packed. These vectors contain different restriction sites, antibiotic resistance markers and

carry large DNA fragments.

3) COSMIDS

It is a hybrid vector derived by cloning plasmids with cos sites of vector. These are similar to

plasmids in many characters such as replication, gene coding, and cleavage sites. These are

dissimilar to it because of presence of DNA fragment from vector. They have small lengths of

about 5Kb.

These were developed by Collins and Hohn in1978.

Cosmids are also used for cloning. This system takes advice of the properties of

both the plasmids and system. A cosmid consist of a circular DNA molecule

containing s plasmid origin of rep. (origin) which allow autonomous replication, a

suitable antibiotic marker, cos site (these are cohesive ends of linear

chromosome infection, the cos site is also required for ligating and pillaging. The

sight amount of DNA into a read).And unique restriction site (e.g. E.Coli) that can

also be used for inserting into DNA fragment. The cosmid can replicate like a

plasmid. The strategy for using the cosmid as a cloning vehicle is illustrated below;

6 | P a g e


7/32

The cosmid can be used to make gene banks by partial endonuclease restriction

(e.g E.Coli) of chromosome that will result in fairly large DNA fragments. These

fragments will be mixed with E.Coli restriction cosmid and joined between 2 linear

cosmid molecule .When the proper size DNA fragment (these can be up to 35 K.blong ) is inserted between 2 cosmid molecule. The distance between the resulting

two cos sites will determine whether the recombinant DNA can be properly

packaged into head to form like particle. When these particle are used to infect

E.Coli. The recombinant cosmid DNA will be injected into the host and replicated by

use of plasmid on present on the molecule proper selection technology will

allow.The detection of specific recombinant DNA molecule in the clones.

4) PHASMIDS

The insertion of plasmid into vector generates a phage genome containing at side and one or

more plasmid molecules. These new genetic combinations are called as phasmids.

PASSENGER DNA

It is DNA which is transferred from one organism into another by combining itself with the

vector DNA.

The three types of DNAs are used as passenger DNA.

i) Complementary DNA (cDNA)

True copy of an mRNA molecule is known as cDNA.It is synthesized on RNA template

with the help of reverse transcriptase enzyme. The DNA strand is isolated from the hybrid

RNA-DNA complex with the use of alkaline phosphatase enzyme. A cDNA strand is then

synthesizes on the isolated single stranded DNA template with the help of DNA polymerase

enzymes. The DNA duplex thus formed can be joined to vector DNA for introduction to a

host cell.

ii) Synthetic DNA

It is synthesizes artificially with the help of DNA polymerases on DNA template.

In 1965, Hargobind Khorana and His associates produce the first artificial DNA without

using a DNA template.

iii) Random DNA

7 | P a g e


8/32

It refers to small fragments produced by breaking a chromosome of an organism with the

help of restriction endonucleases.

Significance of transgenic animals:

1. Increased growth of livestock and fish

2. Increased and improved wool production in sheep

3. Transfection in molecular farming

Application of biotechnology in the field of industry

1. In the area of food production

a) To produce dairy products

i) Milk is pasteurized at 73oC for 15 sec to kill most of the micro-organisms.

ii) Lactobacillus bacteria is used to prepare cheese from milk.

iii) Enzyme rennin is added to separate milk into solid curd and liquid whey.

iv) Ripening of cheese is done by specific microorganisms. It gives flavor to cheese.

v) Yoghurt is prepared from the pasteurized milk with much of the fat removed.For this, special

strain of yoghurt making lactic acid bacteria(lactobacillus bulgaricus and Streptococcus

8 | P a g e


9/32


10/32

This natural ability to alter the plants genetic makeup was the foundation of plant transformation

using Agrobacterium. Currently, Agrobacterium-mediated transformation is the most commonly

used method for plant genetic engineering. One of the reasons for this is the large number ofplants that are susceptible to this bacterium. Initially it was believed that this Agrobacterium only

infects dicotyledonous plants, broadleaf plants, soybeans and tomatoes, for many years, but it

was later established that it can also be used for transformation of monocotyledonous plants suchas rice.

During transformation, several components of the Ti plasmid enable effective transfer of the

genes of interest into the plant cells, these include:

T-DNA border sequences, which demarcate the DNA segment (T-DNA) to be transferred

into the plant genome

Vir genes (virulence genes), which are required for transferring the T-DNA region to the

plant but are not themselves transferred, and

Modified T-DNA region where the genes that cause crown gall formation are removed

and replaced with the genes of interest.

The Agrobacterium-mediated transformation process involves a number of steps:

(a) Isolation of the genes of interest from the source organism;

(b) Insertion of the transgene into the Ti-plasmid;

c) Introduction of the T-DNA-containing-plasmid into Agrobacterium;

d) Mixture of the transformed Agrobacterium with plant cells to allow transfer of T-DNA into

plant chromosome;

(f) Regeneration of the transformed cells into genetically modified (GM) plants; and

(g) Testing for trait performance or transgene expression at lab, greenhouse and field level.

In this method, the tumor inducing (Ti) region is removed from the T-DNA (transfer DNA) and

replaced with the desired gene and a marker, which is then inserted into the organism. The

marker is used to find the organism which has successfully taken up the desired gene. Tissues of

the organism are then transferred to a medium containing an antibiotic or herbicide, dependingon which marker was used. The Agrobacterium present is also killed by the antibiotic. Only

tissues expressing the marker will survive and possess the gene of interest. Thus, subsequentsteps in the process will only use these surviving plants. In order to obtain whole plants from

these tissues, they are grown under controlled environmental conditions in tissue culture. This is

a process of a series of media, each containing nutrients and hormones. Once the plants are

grown and produce seed, the evaluation of the progeny process begins. This process entails

10 | P a g e


11/32

selection of the seeds with the desired traits and then retesting and growing to make sure that the

entire process has been completed successfully with the desired results.

11 | P a g e


12/32

.

12 | P a g e


13/32

Figure: Illustrates Agrobacterium-mediated plant transformation

Advantages And Disadvantages Of Agrobacterium As A Gene Transfer Tool

AdvantagesThe overall advantages of using Agrobacterium-mediated transformation over other

transformation methods are:

1. Technically simple

2. Yields relatively uncomplicated insertion events (low copy number, minimal rearrangements)

3. Unlimited size of foreign DNA

4. efficient (for most plants)5. adaptable to different cell types, culture procedures (protoplasts, tissue sections, non-culture

methods)

6. Transformants are mitotically and meiotically stable7. Intact and stable integration of the transgene (newly introduced gene) into the plant genome

Disadvantages

1. Host range is limited: not all plants may be susceptible to Agrobacterium2. With susceptible plants, accessible culture/regeneration systems must be

adaptable to Agrobacterium-mediated gene transfer

In general, the Agrobacterium method is considered preferable to the gene gun, because of a

greater frequency of single-site insertions of the foreign DNA, which allows for easiermonitoring.

13 | P a g e


14/32


15/32

Plant Transformation Using Particle Bombardment

During early 1990s, it was shown that DNA delivery to plant cells is also possible, when

heavy microparticles (tungsten or gold) coated with the DNA of interest are accelerated to avery high initial velocity (1,400 ft per sec). These microprojectiles, each normally 1-3m in

diameter, are carried by a 'macroprojectile' or the 'bullet' and are accelerated into living plant

cells (target cells can be pollen, cultured cells, cells in differentiated tissues and meristems)so that they can penetrate cell walls of intact tissue. The acceleration is achieved either by an

explosive charge (cordite explosion) or by using shock waves initiated by a high-voltage

electric discharge.The Particle bombardment device, also known as the gene gun, wasdeveloped to enable penetration of the cell wall so that genetic material containing a gene of

interest can be transferred into the cell. Today the gene gun is used for genetic

transformation of many organisms to introduce a diverse range of desirable traits.

Particle bombardment (biolistic) is a physical method widely used for gene transfer into plants.mammals fungi and bacteria.

In this method, 1-2m tungsten or gold particles, coated with the DNA to be used for

transformation, are accelerated to velocities, which enable their entry into plant cells/nuclei.

Particle acceleration is achieved by using a device, which varies considerably in design andfunction.

The most successful devices accelerate particles in one of the two ways:

(1) by using pressurised helium gas or

(2) by the electrostatic energy released by a droplet of water exposed to a high voltage.

The earlier devices used blank cartridges in a modified firing mechanism to provide the energyfor particle acceleration; this is the reason for the name particle gun to this approach. The method

is also called biolistic or ballistic method of DNA delivery.

This technique has general applicability to plant species and can be used to deliver DNA into

virtually all tissues. More importantly, it can be used to transform shoot apical meristems, leaf

blades, immature and mature embryos, mature pollen, and root and shoot sections etc.

Meristematic cells show higher transformation frequency than nondividing cells.

The main components of a helium pressure device are, gas acceleration tube, rupture disc.,stopping screen, macrocarrier carrying particles coated with DNA, and target cells. Thesecomponents are enclosed in a chamber to enable the creation of partial vacuum, which facilitates

particle acceleration and reduces damage to plant cells. After creation of partial vacuum

sufficiently pressurised helium gas is released in the acceleration tube to break the rapture disc.

This generates helium shock waves, which accelerate the macroprojectile to which DNA coatedmicroprojectiles are attached. The macroprojectile is stopped by a stopping screen, and the

15 | P a g e


16/32

microprojectiles pass through this screen and become embedded in the cells kept about 10 mm

below the stopping screen.

Helium is preferable to air since it is lighter and offers certain advantages. Generally a 1000 psi

(pounds per square inch) of helium pressure is used for acceleration.

The macrocarrier or macroprojectile is a 2.5 cm diameter 0.06 mm thick plastic membrane,which is used only once. The microprojectiles, micropartic1es or microcarriers vary in diameter

from 0.5 to 2.0 m; the average size of 1.0 11m is commonly used.

Tungsten particles are cheaper, but are of irregular shape and size, may be toxic to certain cell

types and show surface oxidation which may lead to precipitation of DNA.

Plant transformation using particle bombardment follows the same outline as Agrobacterium-

mediated method. The steps taken include:

1) Isolate the genes of interest from the source organism;

2) Develop a functional transgenic construct including the gene of interest;

promoters to drive expression; codon modification, if needed to increase

successful protein production; and marker genes to facilitate tracking of theintroduced genes in the host plant;

3) Incorporate into a useful plasmid;

4) Introduce the transgenes into plant cells;5) Regenerate the plants cells; and

6) Test trait performance or gene expression at lab, greenhouse and field level.

The particle bombardment method starts with coating tungsten or gold particles(microprojectiles) with plasmid DNA. The coated particles are coated on a macro-projectile,which is accelerated with air pressure and shot into plant tissue on a petri plate, as shown in

Figure 1. A perforated plate is used to stop the macro-projectile, while allowing the

microprojectiles to pass through to the cells on the other side. As the microprojectiles enter thecells, the transgenes are released from the particle surface and may incorporate into the

chromosomal DNA of the cells. Selectable markers are used to identify the cells that take up the

transgene. The transformed plant cells are then regenerated into whole plants using tissueculture.

Particle bombardment also plays an important role in the transformation of organelles such as

chloroplasts, which enables engineering of organelle-encoded herbicide or pesticide resistances

in crop plants and to study photosynthetic processes. Limitations to the particle bombardmentmethod relative to Agrobacterium-mediated transformation include frequent integration of

multiple copies of the transgene at a single insertion site, rearrangement of the inserted genes,

and incorporation of the transgene at multiple insertion sites. These multiple copies can be linked

to silencing of the transgene in subsequent progeny (Yao et al., 2006). Figure 2 and 3 show thedifferent types of gene guns that are currently used in plant transformation.

16 | P a g e


17/32

Figure 1: Diagrammatic illustration of gene transfer using Gene Gun method

Source: http://www.artsci.wustl.edu/~anthro/blurb/fg8.t.gif

Figure 2 Standard Gene Gun

17 | P a g e
http://www.artsci.wustl.edu/~anthro/blurb/fg8.t.gifhttp://www.artsci.wustl.edu/~anthro/blurb/fg8.t.gif


18/32

Figure 3 Helios Gene Gun

Transgenic plants using the above technique have been obtained in many cases

including soybean, tobacco, maize, rice, wheat, etc. Transient expression of genes

transferred in cells by this method has also been observed in onion, maize, rice and

wheat. During 1990s, there was no other gene transfer approach, which met with'

so much ofenthusiasm. Consequently considerable investment was also made in

experimentation and manpower for development ofthis technique. The advantages

ofthis method over microinjection include the following: (i} thousands ofparticles

are accelerated at the same time, causing multiple hits resulting in transfer of

genes into many cells simultaneously; (ii) since intact cells can be used, some of the

difficulties encountered with the use of protoplasts are automatically circumvented;

(iii) the method is universal in its application, so that cell type, size and shape or the

presence/absence of cell walls do not significantly alter its effectiveness.

In view ofthis, particle bombardment method using microprojectiles was used in a

variety of plant species, particularly the cereals. The method was also used

successfully for transfer ofgenes into the plastid genome. However, the emphasis

on 'the use of particle gun decreased towards the end of20th century, due to the

success achieved in cereals with Agrobacterium mediated gene transfer.

18 | P a g e


19/32

Advantages of transgenic plants

1. Improved Nutritional Quality

Milled rice is the staple food for a large fraction of the world's human population. Milling rice

removes the husk and any beta-carotene it contained. Beta-carotene is a precursor to vitamin A.The synthesis of beta-carotene requires a number of enzyme-catalyzed steps. In January 2000, a

group of European researchers reported that they had succeeded in incorporating threetransgenes into rice that enabled the plants to manufacture beta-carotene in their endosperm.

2. Insect Resistance.

Bacillus thuringiensis is a bacterium that is pathogenic for a number of insect

pests. Its lethal effect is mediated by a protein toxin it produces. Through

recombinant DNA methods, the toxin gene can be introduced directly into thegenome of the plant where it is expressed and provides protection against insect

pests of the plant.

3. Disease Resistance.

Genes that provide resistance against plant viruses have been successfully introduced into such

crop plants as tobacco, tomatoes, and potatoes.

19 | P a g e
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Chlorophyll.html#beta_carotenehttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Chlorophyll.html#beta_carotene


20/32

Tomato plants infected

with tobacco mosaic

virus (which attacks

tomato plants as well as

tobacco). The plants in

the back row carry anintroduced gene

conferring resistance to

the virus. The resistant

plants produced three

times as much fruit as

the sensitive plants

(front row) and the same

as control plants.

(Courtesy Monsanto

Company.)

4. Herbicide Resistance.

Questions have been raised about the safety both to humans and to the environment of

some of the broad-leaved weed killers like 2,4-D. Alternatives are available, but they may

damage the crop as well as the weeds growing in it. However, genes for resistance to some of thenewer herbicides have been introduced into some crop plants and enable them to thrive even

when exposed to the weed killer.

Effect of the herbicide bromoxynil on tobacco plants transformed with

a bacterial gene whose product breaks down bromoxynil (top row)

and control plants (bottom row). "Spray blank" plants were treated

with the same spray mixture as the others except the bromoxynil was

20 | P a g e
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/Auxin.html#herbicideshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/Auxin.html#herbicides


21/32

left out. (Courtesy of Calgene, Davis, CA.)

5. Salt Tolerance

A large fraction of the world's irrigated crop land is so laden with salt that it cannot be used togrow most important crops. [Discussion]

However, researchers at the University of California Davis campus have created transgenic

tomatoes that grew well in saline soils. The transgene was a highly-expressed sodium/protonantiport pumpthat sequestered excess sodium in the vacuole of leaf cells. There was no sodium

buildup in the fruit.

6. "Terminator" Genes

This term is used (by opponents of the practice) for transgenes introduced into crop plants to

make them produce sterile seeds (and thus force the farmer to buy fresh seeds for the followingseason rather than saving seeds from the current crop).

The process involves introducing three transgenes into the plant:

A gene encoding a toxin which is lethal to developing seeds but not tomature seeds or the plant. This gene is normally inactive because of a stretchof DNA inserted between it and its promoter.

A gene encoding a recombinase an enzyme that can remove the spacerin the toxin gene thus allowing to be expressed.

A repressor gene whose protein product binds to the promoter of the

recombinase thus keeping it inactive.

How they work

When the seeds are soaked (before their sale) in a solution oftetracycline

synthesis of the repressor is blocked the recombinase gene becomes active the spacer is removed from the toxin gene and it can now be turned on.

Because the toxin does not harm the growing plant only its developing seeds the crop canbe grown normally except that its seeds are sterile.

The use of terminator genes has created much controversy:

Farmers especially those in developing countries want to be able tosave some seed from their crop to plant the next season.

Seed companies want to be able to be able to keep selling seed.

21 | P a g e
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/Soil.html#Desertshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/Diffusion.html#antiporthttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/Diffusion.html#antiporthttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/Diffusion.html#antiporthttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Promoter.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/Antibiotics.html#tetracyclineshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/Soil.html#Desertshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/Diffusion.html#antiporthttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/Diffusion.html#antiporthttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Promoter.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/Antibiotics.html#tetracyclines


22/32

7. Transgenes Encoding Antisense RNA.

These are discussed in a separate page.

Link to it.

8. Biopharmaceuticals

The genes for proteins to be used in human (and animal) medicine can be inserted into plants and

expressed by them.

Advantages:

Glycoproteins can be made (bacteria like E. coli cannot do this). Virtually unlimited amounts can be grown in the field rather than in

expensive fermentation tanks. There is no danger from using mammalian cells and tissue culture medium

that might be contaminated with infectious agents. Purification is often easier.

Corn is the most popular plant for these purposes, but tobacco, tomatoes, potatoes, and rice are

also being used.

Some of the proteins that are being produced by transgenic crop plants:

human growth hormone with the gene inserted into the chloroplast DNA oftobacco plants.

humanized antibodies against such infectious agents as

o HIVo respiratory syncytial virus (RSV)o sperm (a possible contraceptive)o herpes simplex virus, HSV, the cause of "cold sores"

protein antigens to be used in vaccineso An example: patient-specific antilymphoma (a cancer) vaccines. B-cell

lymphomas are clones of malignant B cells expressing on their surfacea unique antibody molecule. Making tobacco plants transgenic for theRNA of the variable (unique) regions of this antibody enables them toproduce the corresponding protein. This can then be incorporated intoa vaccine in the hopes (early trials look promising) of boosting thepatient's immune system especially the cell-mediated branch to

combat the cancer. other useful proteins like lysozyme and trypsin

Application OR Significance of transgenic plants:

1. These can tolerate adverse conditions, such as drought, high temperature frost, salinity

etc.

22 | P a g e
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AntisenseRNA.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/Glycoproteins.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Esch.coli.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Pituitary.html#GHhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Endosymbiosis.html#chloroplasthttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Monoclonals.html#Problems_with_monoclonal_therapyhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AIDS.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BirthControl.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/V/Viruses.html#dsDNAhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CancerImmunotherapy.html#Patient-SpecificCancerVaccineshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BurkittLymphoma.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BurkittLymphoma.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/C.html#clonehttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/B_and_Tcells.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AntigenReceptors.html#antibodieshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CMI.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Enzymes.html#lysozymehttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/GITract.html#pancreashttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AntisenseRNA.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/Glycoproteins.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Esch.coli.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Pituitary.html#GHhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Endosymbiosis.html#chloroplasthttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Monoclonals.html#Problems_with_monoclonal_therapyhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AIDS.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BirthControl.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/V/Viruses.html#dsDNAhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CancerImmunotherapy.html#Patient-SpecificCancerVaccineshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BurkittLymphoma.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BurkittLymphoma.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/C.html#clonehttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/B_and_Tcells.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AntigenReceptors.html#antibodieshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CMI.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Enzymes.html#lysozymehttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/GITract.html#pancreas


23/32

2. These can produce pharmaceutically important compounds such as human insulin,

interferons, hormones, blood clotting factors etc.

3. 1. They have proved to be extremely valuable tools in studies on plantmolecular biology, regulation of gene action, identification ofregulatory/promotary sequences, etc.2. Specific genes have been transferred into plants to improve theiragronomic and other features.3. Genes for resistance to various biotic stresses have been engineered togenerate transgenic plants resistant to insects, viruses, etc.4. Several gene transfers have been aimed at improving the produce quality.

5. Transgenic plants are being used to produce novel biochemicals likehirudin, etc. which are not produced by normal plants.6. Transgenic plants can be used vaccines for immunization againstpathogens; this is fast emerging as an important objective.

Controversies

The introduction of transgenic plants into agriculture has been vigorously opposed by some.

There are a number of issues that worry the opponents. One of them is the potential risk oftransgenes in commercial crops endangering native or nontarget species.

Examples:

A gene for herbicide resistance in, e.g. corn, escaping into a weed speciescould make control of the weed far more difficult.

The gene for Bt toxin expressed in pollen might endanger pollinators likehoneybees.

To date, field studies on Bt cotton and maize (corn) show that the numbers of some nontarget

insects are reduced somewhat but not as much as in fields treated with insecticides.

Another worry is the inadvertent mixing of transgenic crops with nontransgenic food crops.Although this has occurred periodically, there is absolutely no evidence of a threat to human

health.

Despite the controversies, farmers around the world are embracing transgenic crops. Currently in

the United States over 80% of the corn, soybeans, and cotton grown are genetically modified(GM) principally to provide

resistance to the herbicide glyphosate ("Roundup Ready") thus making itpractical to spray the crop with glyphosate to kill weeds without harming thecrop;

resistance to insect attack (by expressing the toxin ofBacillus thuringiensis).

23 | P a g e
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/B.thuringiensis.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/B.thuringiensis.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/B.thuringiensis.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/B.thuringiensis.html


24/32

What are the Advantages and Disadvantages of Transgenic Crops?

The use of transgenic crops has been an issue for many years. Many concerns have been raised

and these generally fall into two categories:

1.A concern about what affect genetically modified material could have on human health. For

example, transgenic crops have been suggested to cause allergies in some people, although it is

uncertain whether transgenic crops are the source of this reaction [3]. Furthermore the antibioticresistance genes placed in these crops has been suggested to cause resistance to antibiotics

leading to super bugs that cannot be killed with antibiotic treatments [3]. The idea of a

population being uncomfortable with ingesting DNA that originated from another source, such as

24 | P a g e


25/32

a virus or bacteria, must also be considered when thinking about producing transgenic crops.

However, to date, there is no evidence of the DNA from transgenic crops being any different

from the DNA ingested from conventional crops.

2. A concern about whether transgenic crops cause damage to the natural environment. One

example includes pollen from transgenic corn, which has been suggested to kill the Monarchbutterfly larvae. It has been shown that hybrid corn expresses a bacterial toxin in its pollen,

which is then dispersed over 60 meters by wind. In this range, the corn pollen is deposited onother plants near cornfields where it can be ingested by non-target organisms including the

monarch butterfly [5]. These butterflies have been found to eat less, have a slower growth rate

and higher death rate [5]. A second example is the hybridization of crops with nearby weeds.This could cause these weeds to attain resistance to herbicides or other things that we have been

trying to avoid for many years. Genes that provide resistance to viral disease or other traits

allowing them to survive in their environment could end up benefiting weed populations arounda crop field. This trait could make that population more difficult to control. To date, there has

been little evidence to support this theory.

On other side of the coin are the notions that support the use of transgenic crops. The potential

benefits of which are quite obvious, including such things as increased yields (to feed a growingpopulation), decreasing the use of pesticides (to save the environment and the cost of pesticides),

and the production of novel crops (such as providing crops with increased nutritional value) [3].

Being able to retrofit any crop to our desires is a powerful concept, especially with the changingclimates of today.

Should We Use Transgenic Crops?

In the end, the perceived advantages and disadvantages of transgenic crops must be married to

each other to provide a crop that is environmentally sound and non-hazardous. Producers oftransgenic crops and the agencies that study their effects are aware of this point. However, todate, there has been little evidence to support either case. More research is required in this field

to determine the true safety of these plants and to decide whether they are safe for both the

environment and for those who consume these products over the ages. At the least, most wouldagree that the potential advantage of producing crops that provide the human population with

more and cheaper food makes transgenic technology a useful invention.

25 | P a g e


26/32

Gene Transfer by Particle Bombardment

ABSTRACT

Biolistic is a short term for biological ballistics; the biolistic process is one by whichbiological molecules, such as DNA and RNA, are accelerated (usually on microcarriers, termed

microprojectiles) by gun powder, compressed gas or other means [1]. In this study, two particle

bombardment system were used to directly transfer two construct which are pUC19 (controlplasmid without P358-GUS) and pIG 121-HM (containing P358-GUS). The fist one is the Helios

Gene Gun from Bio-Rad and the other is the Biolistic PDS-1000/He from Bio-Rad on Arabopsis

thaliana and Nicotina tobacum respectively.

INTRODUCTION

MATERIALS AND METHODS

Plant Material

Arabopsis thaliana plants were grown in peat at 20oC with an 8 hour photoperiod. The 4 to 5

weeks old plants were used for gene delivery bombardment experiments and Nicotiana tabacum

(tobacco) plants were grown at 23oC with a 16 hour photoperiod in vermiculite. When the first

true leaf expanded, the plants were used for the bombardment.

Preparation of DNA-coated gold microcarriers for the Gene Gun system

5 mg gold microcarriers (diameter: 0.6 mm) and 10 ml of 0.05 M spermidine were vortexed for 5

s and sonicated for 10 s in an ultrasonic bath to break up gold clumps. Secondly, 10 mg plasmid

26 | P a g e


27/32

DNA (pUC19 or pIG121 Hm) in 10 ml H2O is added. Then, 10 ml 1 M CaCl2 was added

dropwise while vortexing the mixture at moderate rate. The mixture was incubated for 10 min at

room temperature to precipitate the gold and DNA. After centrifugation for 15 s at 14,000 rpm,the pellet was washed 3 times in 0.1 ml absolute ethanol. The pellet was resuspended in 20 ml

ethanol containing 0.1 mg/ml polyvinylpyrrolidone (PVP). The final volume was adjusted to 0.6ml using the ethanol/PVP solution. The suspension was then used for the cartridge preparation as

follows.

Catridge preparation with the Tubing Prep Station for the Gene Gun system

Firstly, the Gold-Coat tubing was first dried by purging with nitrogen for 15 min. Then, the

suspension was drawn into the Gold-Coat tubing (5 in length) and placed into the Tubing Prep

Station following, microcarriers are allowed to settle for 10 min. Then ethanol then was removedslowly. The Gold-Coat tubing was rotated and the particles were spread onto the inner surface of

the tubing and subsequently dried with a flow of nitrogen. Any unevenly coated sections should

be discarded before the remaining tubing was cut into 0.5 pieces with the Tubing Cutter. Eightcartridges were generated.

Preparation of DNA-coated gold microcarriers for the PDS-1000/He system

Stock suspension of microprojectiles was prepared by mixing 60 mg of 1.0-mm gold particles in

1,000 ml of absolute ethanol which can be stored at -20 oC. Then stock suspension was vortexed

for 30 s and soon after 35 ml of the stock suspension was quickly removed and added to a 1.5 mlmicrocentrifuge tube to be microcentrifuged at high speed for 30 s. After removing ethanol with

micropipette, pellet was resuspended in 1 ml water and microcentrifuged for 5 min. Then,

microprojectiles were resuspended in 25 ml of DNA solution (pUC19 or pIG121-Hm) (1 mg/

ml). After that these reagents were added in written order and at the amount specified: 220 ml ofwater, 250 ml of 2.5 M CaCl2, and 50 ml of 0.1 M spermidine (Sigma Chemical S-0266 stock

solution stored at 20 oC, diluted 14 ml to 1,000 ml with water). Later on, the solution wasmixed thoroughly and vortexed on a vortex shaker for at least 10 min at 4 oC and

microcentrifuged for 5 min and remove supernatant. Resuspension of DNA/microprojectile

precipitate in 600 ml of absolute ethanol by pipetting up and down several times was the next

step before another microcentrifugation for 1 min. Removal of ethanol and resuspension of pelletin 36 ml of absolute ethanol by pipetting up and down until well dispersed then were done before

loading to the macrocarrier sheet. Lastly, 10 ml of the suspension was pipetted as evenly as

possible onto the center of a Mylar macrocarrier sheet. It is important to let the ethanol evaporate.

Each DNA/microprojectile suspension yields enough solution to coat 3 Mylar macrocarriers.

Bombardment Conditions and Transient Gene Expression

Intact leaves of Arabidopsis and tobacco plants were bombarded with the Gene Gun system. Thehelium pressure for Arabidopsis was 75psi

27 | P a g e


28/32

Detached Arabidopsis leaves were bombarded with the PDS-1000/He unit. First, 1,100 psi

rupture disk (that has been soaked with isopropanol) was placed in the unit. The distance between

the rupture disk and macrocarrier was 8-10 mm and between macrocarrier and stopping screen is1 cm. Each Petri dish containing the leaves was placed in the device 6 cm below the stopping

screen. Then chamber was evacuated to 28-29 mm Hg before bombarding the target.

Storage procedure

After bombardment, the leaves were sprayed with water and placed in a growth chamber for 24

hours before assaying for the GUS enzymatic activities. The detached leaves were incubated in amoisturized container.

Histochemical GUS assay

The leaves were submerged in 1 mM 5-bromo-4-chloro-indolyl-b-D-glucuronide (X-Gluc) in abuffered solution [100 mM Na-phosphate buffer (pH 7.0), 10 mM EDTA, 0.5 mM K3Fe(CN)6,

0.5 mM K4Fe(CN)6, 1.0 mM X-Gluc, 0,1% Triton X-100] and incubated in the dark for 16 hours

at 37 oC.

After staining, chlorophyll was removed with absolute ethanol in order to better visualize the

blue spots.

RESULTS:

Figure 1: Gene gun (pIG121 Hm)

28 | P a g e


29/32


30/32

promoter whereas, pUC19 does not (negative control).

DISCUSSION

Despite, Argobacterium based based transformation widely used; it is almost but exclusively

restricted to dicotyledons [9] Since, monocotyledonous plants, which constitute some of theworld's most important food crops, there is an alternative method is required. Microprojectile

bombardment, which is non-specific, provides an ideal alternative approach and has been

successfully employed to transform several of the major cereals, including barley maize wheatrice together with other monocotyledons such as tulip and orchids [13].Unlike the other direct

transformation methods, the Microprojectile bombardment permits the delivery of DNA to the

wide range of plants (review), making it the choice of method used in transformation.

There are several parameters that affect the efficiency of this method. These include the

properties of microprojectile, tissue culture method of the plants, DNA construct and etc.

Nature of Microprojectiles

The two major microprojectiles or microcarriers are gold and tungsten particles. The size of the

particle is important and should be optimized in such a way that can deliver significant amount ofDNA without causing damage. Beside this the spherical shape is advantageous as it gives less

damage to the cell. In general, tungsten particles have an irregular surface and are predisposed to

agglomeration during the DNA coating procedure, whereas gold microprojectiles, which are

nearly spherical, remain separated [10].Moreover, these particles should be inert materialsotherwise they may cause toxicity and degradation of DNA in the cell. The gold particles are not

toxic to any of the cells assessed by Sanford et al. [11] and did not catalytically attack DNAadsorbed to the surface of particles where as, tungsten is toxic to certain cell types and is also

subject to surface oxidation, which affects DNA binding and degradation of adhered DNA[13].

Finally, the concentrations of these carriers are another parameter to be considered. If the

concentration too

Low, it will give sparse coverage over the target area while high concentrations of

microprojectiles can result in agglomeration of the particles during the DNA coating procedure.

Agglomerated particles can lead to excessive damage to plant tissues upon bombardment

Adherence of DNA to microprojectiles

No doubt that the efficient DNA coatings of microprojectiles are essential for optimal delivery tothe target. In both two procedures CaCl2 was used to precipitate DNA with gold particles.

Polyvinyllpyrrolidone (PVP) was used as an adhesive during the coating of DNA/microcarrier

suspension to the walls of the Goldcoat tubing[bulletin], resulting increased evenly coating. Andthe spermidine which is a polyamide was used to increase static charges on plasmid GC rich

30 | P a g e


31/32

regions hence increasing the coating efficiency. Sonification was done to disrupt any aggregated

particles. Resuspension of DNA/microprojectile with ethanol permits the precipitate to dry as

ethanol evaporates easily.

Uneven precipitation of DNA and the aggregation of microprojectiles are the problemsencountered during the coating of DNA/microprojectiles. Uneven precipitation is due to rapid

process of precipitation itself. The vortexing procedure is commonly used while adding the

calcium chloride drop wise to the DNA microprojectile mixture.

Increasing the concentration of DNA used to coat the microprojectiles should, theoretically,increase the transformation frequency in a linear manner, until saturation of the microprojectiles

is achieved. However, high concentrations of DNA have been shown to result in agglutination of

the

microprojectiles, which reduce transformation frequencies because of the large size and,

effectively, the reduced number of particles [12].

Delivery of Microprojectiles into Target Tissues

Accelerating power, distance between device and the target and the vacuum in PDS-100/He

system are important factors for velocity and so for penetration. Most systems utilize a vacuum toreduce air drag on the microprojectiles and to reduce the risk of shock waves, from the overlying

gas, damaging the tissue [11]. Vacuum mediates a uniform distribution of particles.

Incorporation of Introduced DNA into the Recipient Genome

Integration of the DNA construct into the chromosome results in stable expression of thetransgene where as otherwise, it will give a transient expression or most probably nothing. In aclearer sentence, the gene expression is related to the ultimate location of microprojectiles within

cells.

Choice of DNA Construct

The choice is extremely dependent on the purpose of the experiment itself. In this

study, the purpose was to observe the succession of the two different gene

bombardment procedures by using a reporter enzyme GUS. The GUS enzyme is

encoded by an E coli gene (gusA or udiA). GUS (-Glucoronidase) cleaves a

colourless substrate, X-Gluc (5 bromo-4-chlom-3-indolyl-l~-D-glucoronic acid) into aproduct which, on oxidation, produces an indigo coloured dye. Hence, the

transformed cells can be identified by their blue colouration [13].

The DNA construct pIG121Hm that was used contains this enzyme with a promoter

called Cauliflower Mosaic Virus (CaMV) 35S promoter where as the DNA construct

pUC19 does not contain the gene, used for negative control. The CaMV 35S

promoter is very strong and seemed to be constitutive, meaning inducing the

31 | P a g e


32/32

expression of the downstream located coding region, in apparently all plant tissues

[1]

Configuration of the vector used for gene delivery may influence gene integration

and expression as linear forms of plasmid resulted in higher levels of gene

expression than supercoiled DNA [13].

Constructs with selectable marker which is used to select the transformed cells to

get a new plant can be used with a transgene of interest. The selectable marker and

transgene may have their own promoters one is constitutive and the other is

development or tissue specific depends on the interest opening an amazing doors of

the science for use of the humans.

Choice and Preparation of Explants

In the experiment the leaves used were young allowing easy penetration for the bombardment.

r DNA Tchnology

Documents

Transcript of r DNA Tchnology