genetic engineering technologies

7/30/2019 Genetic Engineering Technologies

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Genetic engineering technologies

Introduction

Genetic engineering, also called genetic modification, is the direct manipulation of an

organism's genome using biotechnology. New DNA may be inserted in the host genome by

first isolating and copying the genetic material of interest using molecular cloning methods to

generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into

the host organism. Genes may be removed, or "knocked out", using a nuclease. Gene

targeting is a different technique that uses homologous recombination to change an

endogenous gene, and can be used to delete a gene, remove exons, add a gene, or introduce

point mutations. An organism that is generated through genetic engineering is considered to

be a genetically modified organism (GMO). The first GMOs were bacteria in 1973; GM mice

were generated in 1974. Insulin-producing bacteria were commercialized in 1982 and

genetically modified food has been sold since 1994.

Genetic engineering techniques have been applied in numerous fields including research,

agriculture, industrial biotechnology, and medicine. Enzymes used in laundry detergent and

medicines such as insulin and human growth hormone are now manufactured in GM cells,

experimental GM cell lines and GM animals such as mice or zebrafish are being used for

research purposes, and genetically modified crops have been commercialized.

Genetic engineering alters the genetic makeup of an organism using techniques that

remove heritable material or that introduce DNA prepared outside the organism either

directly into the host or into a cell that is then fused or hybridized with the host. This involves

using recombinant nucleic acid (DNA or RNA) techniques to form new combinations of

heritable genetic material followed by the incorporation of that material either indirectly

through a vector system or directly through micro-injection, macro-injection and micro-

encapsulation techniques.Genetic engineering does not normally include traditional animal and plant breeding, in

vitro fertilisation, induction of polyploidy, mutagenesis and cell fusion techniques that do not

use recombinant nucleic acids or a genetically modified organism in the process. However

the European Commission has also defined genetic engineering broadly as including selective

breeding and other means of artificial selection. Cloning and stem cell research, although not

considered genetic engineering are closely related and genetic engineering can be used within

them. Synthetic biology is an emerging discipline that takes genetic engineering a step further

by introducing artificially synthesized genetic material from raw materials into an organism.


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If genetic material from another species is added to the host, the resulting organism is called

transgenic. If genetic material from the same species or a species that can naturally breed

with the host is used the resulting organism is called cisgenic. Genetic engineering can also

be used to remove genetic material from the target organism, creating a gene knockout

organism. In Europe genetic modification is synonymous with genetic engineering while

within the United States of America it can also refer to conventional breeding methods. The

Canadian regulatory system is based on whether a product has novel features regardless of

method of origin. In other words, a product is regulated as genetically modified if it carries

some trait not previously found in the species whether it was generated using traditional

breeding methods (e.g selective breeding, cell fusion, mutation breeding) or genetic

engineering. Within the scientific community, the term genetic engineering is not commonly

used; more specific terms such as transgenic are preferred.

History:-

Genetic engineering as the direct manipulation of DNA by humans outside breeding and

mutations has only existed since the 1970s. The term "genetic engineering" was first coined

by Jack Williamson in his science fiction novel Dragon's Island, published in 1951, one year

before DNA's role in heredity was confirmed by Alfred Hershey and Martha Chase and two

years before James Watson and Francis Crick showed that the DNA molecule has a double-

helix structure. In 1974 Rudolf Jaenisch created the first GM animal.

In 1972 Paul Berg created the first recombinant DNA molecules by combining DNA from the

monkey virus SV40 with that of the lambda virus. In 1973 Herbert Boyer and Stanley Cohen

created the first transgenic organism by inserting antibiotic resistance genes into the plasmid

of an E. coli bacterium. A year later Rudolf Jaenisch created a transgenic mouse by

introducing foreign DNA into its embryo, making it the worlds first transgenic animal.

These achievements led to concerns in the scientific community about potential risks from

genetic engineering, which were first discussed in depth at the Asilomar Conference in 1975.

One of the main recommendations from this meeting was that government oversight of

recombinant DNA research should be established until the technology was deemed safe.

In 1976 Genentech, the first genetic engineering company was founded by Herbert Boyer and

Robert Swanson and a year later and the company produced a human protein (somatostatin)

in E.coli. Genentech announced the production of genetically engineered human insulin in

1978. In 1980, the U.S. Supreme Court in the Diamond v. Chakrabarty case ruled that


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genetically altered life could be patented. The insulin produced by bacteria, branded humulin,

was approved for release by the Food and Drug Administration in 1982.

In the 1970s graduate student Stephen Lindow of the University of WisconsinMadison with

D.C. Arny and C. Upper found a bacterium he identified as P. syringae that played a role in

ice nucleation and in 1977, he discovered a mutant ice-minus strain. He was later successful

at created a recombinant ice-minus strain. In 1983, a biotech company, Advanced Genetic

Sciences (AGS) applied for U.S. government authorization to perform field tests with the ice-

minus strain of P. syringae to protect crops from frost, but environmental groups and

protestors delayed the field tests for four years with legal challenges. In 1987, the ice-minus

strain of P. syringae became the first genetically modified organism (GMO) to be released

into the environment when a strawberry field and a potato field in California were sprayed

with it. Both test fields were attacked by activist groups the night before the tests occurred:

"The world's first trial site attracted the world's first field trasher".

The first field trials of genetically engineered plants occurred in France and the USA in 1986,

tobacco plants were engineered to be resistant to herbicides. The Peoples Republic of China

was the first country to commercialize transgenic plants, introducing a virus-resistant tobacco

in 1992. In 1994 Calgene attained approval to commercially release the Flavr Savr tomato, a

tomato engineered to have a longer shelf life. In 1994, the European Union approved tobacco

engineered to be resistant to the herbicide bromoxynil, making it the first genetically

engineered crop commercialized in Europe. In 1995, Bt Potato was approved safe by the

Environmental Protection Agency, after having been approved by the FDA, making it the

first pesticide producing crop to be approved in the USA. In 2009 11 transgenic crops were

grown commercially in 25 countries, the largest of which by area grown were the USA,

Brazil, Argentina, India, Canada, China, Paraguay and South Africa.

In the late 1980s and early 1990s, guidance on assessing the safety of genetically engineered

plants and food emerged from organizations including the FAO and WHO.

In 2010, scientists at the J. Craig Venter Institute announced that they had created the first

synthetic bacterial genome and added it to a cell containing no DNA. The resulting

bacterium, named Synthia, was the world's first synthetic life form.


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GENE TRANSFER TECHNOLOGIES

Gene transfer technology provides the ability to genetically manipulate the cells of higher

animals. During the 1970s it became possible to introduce exogenous DNA constructs into

higher eukaryotic cells in vitro. Mammalian (germline) transgenesis was first achieved in the

early 1980s. The model used in this study was mice. The delivery of genes in vitro can be

done by treating the cells with viruses such as retrovirus or adenovirus, calcium phosphate,

liposomes, particle bombardment, fine needle naked DNA injection, electroporation or any

combination of these methods. These are the powerful tools for research and have possible

applications in gene therapy. A number of valuable techniques used to transfer genes in

animals and plants cells and their scope and contributions are explained below.

DNA transfer by artificial methods & Physical methods1. Macroinjection2. Microinjection3. Protoplast fusion4. Biolistics transformation1. Chemical methods1. DNA transfer by calcium phosphate method2. Transfer of DNA by use of polyethene glycol3. Use of DEAE-Dextran for DNA transfer4. Liposome mediated transfer2. Electrical methods1. Electroporation2. Electrofusion3. Electroporation4. Electroporation uses electrical pulse to produce transient pores in the plasma

membrane thereby allowing macromolecules into the cells. It is an efficient process to

transfer DNA into cells. Microscopic pores are induced in biological membrane by

the application of electric field. These pores are known as electropores which allow

the molecules, ions and water to pass from one side of the membrane to another. The

pores can be recovered only if a suitable electric pulse is applied. The electropores

reseal spontaneously and the cell can recover. The formation of electropores depends

upon the cells that are used and the amplitude and duration of the electric pulse that is

applied to them. Electric currents can lead to dramatic heating of the cells that can


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results in cell death. Heating effects are minimized by using relatively high amplitude,

a short duration pulse or by using two very short duration pulses. In terms of

mammalian transgenesis, electroporation is an effective method of introducing

exogenous DNA into embryonic stem (ES) cells. This technique has recently, been

used to transfer genes into cultured mammalian embryos at defined stages of

development. It was reported that there is an increase from 12 to 19% of transgenic

bovine blastocysts when electroporation was included in an otherwise passive sperm-

DNA uptake protocol. Similar findings were reported again with transgenic bovine

blastocysts. Fish species were also reported to be genetically manipulated.

Electroporation has been reported to enhance the level of gene expression and

significantly improve immune responses elicited to DNA vaccines in both large and

small animals.

General applications of electroporation

1. Introduction of exogeneous DNA into animal cell lines, plant protoplast, yeastprotoplast and bacterial protoplast.

2. Electroporation can be used to increase efficiency of transformation or transfection ofbacterial cells.

3. Wheat, rice, maize, tobacco have been stably transformed with frequency upto 1% bythis method.

4. Genes encoding selectable marker may be used to introduce genes usingelectroporation.

5. To study the transient expression of molecular constructs.6. Electroporation of early embryo may result in the production of transgenic animals.7. Hepatocytes, epidermal cells, haematopoietic stem cells, fibroblast, mouse T and B

lymphocytes can be transformed by this technique.

8. Naked DNA may be used for gene therapy by applying electroporation device onanimal cells.

Advantages of electroporation

1. Method is fast.2. Less costly.3. Applied for a number of cell types.4. Simultaneously a large number of cell can be treated.5. High percentage of stable transformants can be produced.


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Microinjection

In microinjection DNA can be introduced into cells or protoplast with the help of very fine

needles or glass micropipettes having the diamete

injected may be taken up by the nucleus. Computerized control of holding pipette, needle,

microscope stage and video technology has improved the efficiency of this technique. The

advantages, limitations and applications are listed in the Table 5, 6 and 7 respectively.

Advantages of microinjection

1. Frequency of stable integration of DNA is far better as compare to other methods.2. Method is effective in transforming primary cells as well as cells in established

cultures.

3. The DNA injected in this process is subjected to less extensive modifications.4. Mere precise integration of recombinant gene in limited copy number can be

obtained.

Limitations of microinjection

1. Costly.2. Skilled personal required.3. More useful for animal cells.4. Embryonic cells preferred for manipulation.5. Knowledge of mating timing, oocyte recovery is essential.6. Method is useful for protoplasts and not for the walled cells.7. Process causes random integration.8. Rearrangement or deletion of host. DNA adjacent to site of integration are common.

Applications of microinjection

1. Process is applicable for plant cell as well as animal cell but more common for animalcells.

2. Technique is ideally useful for producing transgenic animal quickly.3. Procedure is important for gene transfer to embryonic cells.4. Applied to inject DNA into plant nuclei.5. Method has been successfully used with cells and protoplast of tobacco, alfalfa etc.

Microinjection is potentially a useful method for simultaneous introduction of multiple

bioactive compounds such as antibodies peptides RNAs plasmids diffusion markers elicitors

Ca2+

as well as nucleus and artificial micro or nanoparticles containing those chemicals into

the same target single-cells.


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Macroinjection

Macroinjection is the method tried for artificial DNA transfer to cereals plants that show

inability to regenerate and develop into whole plants from cultured cells. Needles used for

injecting DNA are with the diameter greater than cell diameter. DNA injected with

conventional syringe into region of plant which will develop into floral tillers. Around 0.3 ml

of DNA solution is injected at a point above tiller node until several drops of solution came

out from top of young inflorescence. Timing of injection is important and should be

fourteen days before meiosis. This method was found to be successful with rye plants. It is

also being attempted for other cereals plants.

Advantages and limitations of macroinjection

1. This technique does not require protoplast.2. Instrument will be simple and cheap.3. Methods may prove useful for gene transfer into cereals which do not regenerate from

cultured cell easily.

4. Technically simple.Limitations

1. Less specific.2. Less efficient.3. Frequency of transformation is very low.

Biolistics or microprojectiles for DNA transfer

Biolistics or particle bombardment is a physical method that uses accelerated microprojectiles

to deliver DNA or other molecules into intact tissues and cells. The method was developed

initially to transfer genes into plants by Sanford. Ballistics transformation is relatively new

and novel method amongst the physical methods for artificial transfer of exogenous DNA.

This method avoids the need of protoplast and is better in efficiency. This technique can be

used for any plant cells, root section, embryos, seeds and pollen. The gene gun is a device

that literally fires DNA into target cells. The DNA to be transformed into the cells is coated

onto microscopic beads made of either gold or tungsten. Beads are carefully coated with

DNA. The coated beads are then attached to the end of the plastic bullet and loaded into the

firing chamber of the gene gun. An explosive force fires the bullet down the barrel of the gun

towards the target cells that lie just beyond the end of the barrel. When the bullet reaches the

end of the barrel it is caught and stopped, but the DNA coated beads continue on toward the

target cells. Some of the beads pass through the cell wall into the cytoplasm of the target

cells. Here the bead and the DNA dissociate and the cells become transformed. Once inside


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the target cells, the DNA is solubilised and may be expressed. This approach, sometimes

known as biolistics, was originally developed for plant transgenesis but has been shown to

be effective for transferring transgenes into mammalian cells in vivo. The gene gun is

particularly useful for transforming cells that are difficult to tran , e.g. plant cells. It is also

gaining in use as a method for transferring DNA construct into whole animals.

General applications of biolistics

1. Biolistics technique has been used successfully to transform soyabean, cotton, spruce,sugarcane, papaya, corn, sunflower, rice, maize, wheat, tobacco etc.

2. Genomes of subcellular organelles have been accessible to genetic manipulation bybiolistic method.

3. Mitochondria of plants and chloroplast of chlamydomonas have been transformed.4. Method can be applied to filamentous fungi and yeast (mitochondria).5. The particle gun has also been used with pollen, early stage embryoids, meristems and

somatic embryos.

Advantages and limitations of biolistics

Advantages

1. Requirement of protoplast can be avoided.2. Walled intact cells can be penetrated.3. Manipulation of genome of subcellular organelles can be achieved.

Limitations

1. Integration is random.2. Requirement of equipments.

Liposome mediated gene transfer

Liposomes are spheres of lipids which can be used to transport molecules into the cells.

These are artificial vesicles that can act as delivery agents for exogenous materials including

transgenes. They are considered as sphere of lipid bilayers surrounding the molecule to be

transported and promote transport after fusing with the cell membrane. Cationic lipids are

those having a positive charge are used for the transfer of nucleic acid. These liposomes are

able to interact with the negatively charged cell membrane more readily than uncharged

liposomes, with the fusion between cationic liposome and the cell surface resulting in the

delivery of the DNA directly across the plasma membrane. Cationic liposomes can be

produced from a number of cationic lipids, e.g. DOTAP (N-1(2,3-dioleoyloxy) propyl)-

N,N,N-trimethylammoniumethyl sulphate) and DOTMA (N-(1-(2,3-dioleoyloxy)propyl)-


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N,N,N-trimethylammonium chloride) [34]. These are commercially available lipids that are

sold as an in vitro-transfecting agent, as lipofectin.

Advantages of liposome mediated DNA transfer

Advantages

1. Simplicity.2. Long term stability.3. Low toxicity.4. Protection of nucleic acid from degradation.

Liposomes for use as gene transfer vehicles are prepared by adding an appropriate mix of

bilayer constituents to an aqueous solution of DNA molecules. In this aqueous environment,

phospholipid hydrophilic heads associate with water while hydrophobic tails self-associate to

exclude water from within the lipid bilayer.Bilayer membrane enveloping a small quantity of

DNA solution. The liposomes are then ready to be added to target cells. Germline

transgenesis is possible with liposome mediated gene transfer and ES cells have been

successfully transfected by liposomes also.

Calcium phosphate mediated DNA transfer

The process of transfection involves the admixture of isolated DNA (10-100ug) with solution

of calcium chloride and potassium phosphate under condition which allow the precipitate of

calcium phosphate to be formed. Cells are then incubated with precipitated DNA either in

solution or in tissue culture dish. A fraction of cells will take up the calcium phosphate DNA

precipitate by endocytosis. Transfection efficiencies using calcium phosphate can be quite

low, in the range of 1-2 %. It can be increased if very high purity DNA is used and the

precipitate allowed to form slowly. Procedures have been developed where cell taking up

exogenous DNA could be upto 20%. This technique is used for introducing DNA into

mammalian cells.

Limitations of calcium phosphate mediated DNA transfer

Limitations

1. Frequency is very low.2. Integrated genes undergo substantial modification.3. Many cells do not like having the solid precipitate adhering to them and the surface of

their culture vessel.

4. Integration with host cell chromosome is random.5. 5, Due to above limitations transfection applied to somatic gene therapy is limited.


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DNA transfer by DAE-Dextran method

DNA can be transferred with the help of DAE Dextran also. DAE-Dextran may be used in

the transfection medium in which DNA is present. This is polycationic, high molecular

weight substance and convenient for transient assays in cos cells. It does not appear to be

efficient for the production of stable transfectants. If DEAE-Dextran treatment is coupled

with Dimethyl Sulphoxide (DMSO) shock, then upto 80% transformed cell can express the

transferred gene. It is known that serum inhibits this transfection so cells are washed nicely to

make it serum free. Stable expression is very difficult to obtain by this method. Treatment

with chloroquinine increases transient expression of DNA. The advantage of this method is

that, it is cheap, simple and can be used for transient cells which cannot survive even short

exposure of calcium phosphate.

Transfer of DNA by polycation-DMSO As calcium phosphate method of DNA transfer is

reproducible and efficient but there is narrow range of optimum conditions. DNA transfer by

polycation, polybrene is used to increase the adsorption of DNA to the cell surface followed

by a brief treatment with 25-30% DMSO to increase membrane permeability and enhance

uptake of DNA. In this method no carrier DNA is required and stable transformants are

produced. This method works with mouse fibroblast and chick embryo.

Polyethylene glycol mediated transfection

This method is utilized for protoplast only. Polyethylene glycol stimulates endocytosis and

therefore DNA uptake occurs. Protoplasts are kept in the solution containing polyethylene

glycol (PEG). The molecular weight of PEG used is 8000 dalton having the final

concentration of 15%. Calcium chloride is added and sucrose and glucose acts as osmotic

buffering agent. To reduce the effects of nuclease present, the carrier DNA from salmon or

herring sperm may also be added. After exposure of the protoplast to exogenous DNA in

presence of PEG and other chemicals, PEG is allowed to get removed. Intact surviving

protoplasts are then cultured to form cells with walls and colonies in turn. After several

passages in selectable medium frequency of transformation is calculated. PEG based vehicles

were less toxic and more resistant to nonspecific protein adsorption making them an

attractive alternative for nonviral gene delivery. PEG PBLG nanoparticle mediated HSV

TK/GCV gene therapy for oral squamous cell carcinoma was also reported.

Gene transfer through peptide

A variety of peptide sequences are there which are able to bind to, and condense, DNA to

make it more amenable for entry into cells; e.g. the tetrapeptide Serine - proline-lysine-

lysine (present on the C- terminus of the histone H1 protein) helps in DNA transfer [44].


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Lysine is a positively charged amino acid. The positively charged lysine amino acid side

chains help to counteract the negatively charged phosphate DNA backbone and allow the

DNA molecules to pack closely to each other. Rational design of peptide sequences has also

been used to develop synthetic DNA binding peptide. Tyrosine-lysine-alanine-(Lysine)s-

tryptophan-lysine is a peptide which is very effective to form complexes with DNA. DNA

binding peptides that can be coupled to cell specific ligands can also be synthesized. It allows

receptor mediated targeting of the peptide/DNA complexes to specific cell types.

Gene transfer by retroviruses

The relatively low efficiency of foreign DNA integration into animal cells, combined with the

lack of naturally occurring plasmids, led to the manipulation of viruses as potential vectors

for gene transfer. Retroviruses are found in many species including most mammals. The

genome of retroviruses can be manipulated to carry exogenous DNA. The advantages of

using retrovirus based vectors arise from the stability of the integration of the viral genome

into the host. The integration of a single copy of the viral DNA at a random location within

the hosts genome allows for the long term expression of the integrated foreign gene.

Additionally, retroviruses represent a highly efficient mechanism for the transfer of DNA into

cells. Virus uptake is effective for many somatic cell lines, however germline cells are

infected at low frequency, due to a high level of mosaicism. Virus can be used for highly

developed tissues, such as those of foetuses, juveniles or adults. This holds great promise in

the context of somatic gene therapy. Retroviral vectors have also been used to introduce

transgenes into the ES cell genome. However, retroviral vectors are limited or problematic in

a number of respects. They exhibit random nature of integration process, which may have

deleterious effects on the host cell, and the general requirement that retrovirus have to infect

only dividing cells.

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