1GENETIC ENGINEERING

DNA (GENE) LIBRARYA gene library is the collection of different DNA sequences from an organism where each sequence has been cloned into a vector for ease of purification, storage and analysis. There are two types of libraries on the basis of source of DNA used:

(i) genomic library (where genomic DNA is used), and (ii) cDNA library (where cDNA or complementary DNA is produced using mRNA).

A gene library should contain a certain number of recombinants with a high probability of containing any particular sequence. This value can be calculated if you know the genome sizeGenomic LibraryIt is a collection of clones that represent the complete genome of an organism. All fragments of DNA inserted into vectors for further propagation into suitable host represents the entire genome of an organism. For construction of a genomic library the entire genomic DNA is Isolated from host cells/tissues, purified and broken randomly into fragments of correct size for cloning into a suitable vector. There are two basic ways of fragmenting genomic DNA randomly: physical shearing (e.g. pipetting, mixing or sonication) and partial restriction enzyme digestion (by using limiting amount of restriction enzyme the DNA is not digested at every recognition sequence). Using these methods genomic DNA is broken randomly into smaller fragments.Following Fig. shows a total of 6 random DNA fragments obtained after physical shearing. vector isolated from bacterium is also digested with the restriction enzyme which digests genomic DNA. The fragments of genomic DNA is inserted into the vector. Each vector consists of diff of DNA. The recombinant DNA molecules are transferred into bacterial cells bacteriophage particles are assembled.The genomic library for organisms with smaller genome size (e.g. E. coli) can be construe, in plasmid vector. Only 5,000 clones (of the average size of 5,000 bp) results in 99% chance cloning the entire genome of 4.6 x 106 bp. In addition, libraries from organisms with larger genomes are constructed using phase λ, cosmid, BAC or YAC vectors.

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cDNA LibraryThe mRNAs are highly processed representatives of genes which express under specific conditions. The mRNAs cannot be directly cloned because they are unstable hence mRNAs are converted into cDNAs. The library made from complementary or copy DNA (cDNA) is called cDNA library. The cDNA library represents the DNA of only eukaryotic organisms, not the prokaryotic ones. Because genomic DNA of eukaryotes contains introns (A gene segment that is transcribed from DNA which later on is excised from the primary transcript during processing to a functional RNA molecule and Exon is the coding region of the gene that

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3are present in processed mRNA), regulatory regions, repetitive sequences. Therefore, establishment of genomic library of eukaryotes is not meaningful. The cDNA library can be constructed by using mRNA because mRNAs are the highly processed, intron-free representatives of DNA having only coding sequence. Keeping in view the aim of the experiment, DNA molecule is either enzymatically fragmented or maybe procured from cDNA or genomic libraries. Getting a cDNA fragment of its Known function or characterizing its sequence is very difficult. A cDNA fragment is prepared directly by using mRNA as template. It follows many biochemical methods.r-DNA and Gene CloningThe hybrid DNA produced by joining pieces of DNA at specific point is known as r-DNA or recombinant DNA.Insertion of a gene into a vector to form a new DNA molecule that can be perpetuated into host cell is known as gene cloning or molecular cloning.The DNA fragment to be introduced into the host cell is called insert DNA, desired DNA, target DNA or foreign DNA. Such gene/DNA fragment is taken out from an organism or gene library i.e. a random collection of cloned DNA fragments in vectors that consist of all genetic information of that organism concerned. However, getting a desired DNA is not so easy because the cellular DNA is very large and the position of desired DNA is not accurately known. Libraries of such cloned fragments are prepared.

The principal steps of gene cloning are shown in the following Fig. and given below:

01. Isolation of DNA of known function from an organism (A).02. Enzymatic cleavage (B) and joining (C) of insert DNA to another DNA molecule

(cloning vector) to form a recombinant DNA (i.e. vector + insert DNA) molecule (D).

03. Transformation of a host cell i.e. transfer and maintenance of this rDNA molecule into a host cell (E).

04. Identification of transformed cells (i.e. cells carrying rDNA) and their selection from non-transformants.

05. Amplification of rDNA (F) to get its multiple copies in a cell.06. Cell multiplication (G) to get a clone i.e. a population of genetically identical cells.

This facilitates each of clones to possess multiple copies of foreign DNA.

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Transgene : A gene from one source incorporated into the genome of another organism.

Transgenesis: Introduction of a gene into animal or plant cells that is successively transmitted to onward generation.

Transgenic Animal: A fertile animal consisting of a foreign gene in its germ line (the cells that produce gamete).

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TRANSGENESIS: Modification of germ cells by gene manipulation in vitro is called transgenesis/ transfection/ transformation.TRANSGENIC ANIMAL: Animal carrying new gene(s) called transgenic.Particular variants of species into which a new gene(s) has been inserted.Every cell of the animal carries new genetic information within the genome.Why Transformation is Performed?

1. To incorporate trait to enhance the conventional animal product2. To incorporate traits for biosynthesis of new product3. Study of gene expression in mammalian development4. Study of tissue specific expression5. Study of genetic basis of various diseases6. Gene function study through knockouts7. Minimize the time requirement to introduce a trait that is needed in breeding

Methods of Introducing Exogenous DNA Into Mammalian Cells1. Nuclear transformation2. DNA/ Calcium phosphate co-precipitate method3. Microinjection of eggs4. Electroporation 5. Use of viral vectors6. Lipofection

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6Nuclear Transformation

Microinjection of Eggs

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Applicable to large cells like oocytes, eggs, early embryo

Efficiency of Producing Transgenic Animals by Microinjection

Animal Animal SpeciesSpecies

No. of ova No. of ova injectedinjected

No. of No. of offspringoffspring

No. of transgenic No. of transgenic offspringoffspring

RabbitRabbit 19071907 218218 2828

SheepSheep 10321032 7373 11

PigPig 20352035 192192 2020

Electroporation

Step I: Cells are exposed to a brief pulse of electricityStep II: Generation of transient, nanometre-sized pores in the cell membrane.Step III: DNA enters through the pores and is transported to the nucleus.First applied to mouse fibroblast by Wong and Neumann (1982).

Factors:Intensity and duration of the electric pulse

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1. Microinjection of DNA (2 pl of buffer containing cloned DNA) into male pro-nucleus of the newly fertilized egg.

2. Two pronuclei fuse to form diploid zygote nucleus of the fertilized egg.

3. Implantation manipulated ova in a pseudopregnant foster mother.

8Disadvantages:High input costLarge number of cell requires (50% cell death occur in the most efficient electroporation)

Transgenic Mice Production

Application of Transgenic Mice

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91. Understanding gene regulation2. Immunological specificity understanding and antibody development3. Drug production4. Onset and progress of human disease e.g. Alzheimer disease, arthritis, muscular

dystrophy, tumorigenesis etc.Transgenic Cattle

Application of Transgenic Cattle

1. Use as bioreactor (mammary gland, product as secretary compound). E.g. protein C (preventing blood clot),

factor IX (facilitate blood clot)κ-casein production (cheese production)lactase transgene

2. Resistance towards bacterial, viral and parasitic diseases by introducing transgene or through in vivo immunization. (thus cost involved with vaccination which contribute 20% of the production and that cost can be reduced through in vivo immunization).

What is gene therapy?

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Collection of oocytesIn vitro maturation of oocytes

In vitro fertilizationCentrifugation of egg to concentrate yolkDNA microinjection onto male pronuclei

In vitro embryonic development to blastocyst stageNon-surgical embryo implantation into recipient foster mother

Screening of offspring for transgene

2 transgenics from 2,470 oocytes. Feasible by not inefficient technique.

10Introduction of a fully functional and expressible gene into a target cell, with the result that a specific genetic disease Will be corrected permanently. Gene therapy provides a functional form of a protein which is either lacking or defective, or reduce expression of any gene which is causing deleterious effect on human.

Why gene therapy is needed?

Gene has many functions and mutation may cause1. Accumulation of a substrate that is toxic for the body2. Deficiency of a compound that is vital for cellular operations3. Mutation in structural gene causing severe architectural abnormalities4. defect in a gene that is expressed in one tissue may have impact on other tissue.

Correction of these genetic defect is absolutely necessary. If not then in short time the patient dies.

Type of Gene Therapy:

1. Ex vivo gene therapy2. In vivo gene therapy

1. Ex vivo gene therapy

Transfection of the patients defective cell out side the body.2. In vivo gene therapy

Direct delivery of the remedial gene into the defective cells of a particular tissue of a prospective patient

Genetic Diseases

Phenylketonuria

A genetic disease in Caucasian newborns. They have mutation on the gene for the liver enzyme phenylalanine dehydroxylase which prevents the conversion of the amino acid phenylalanine to tyrosin. This conversion finally effect on nervous tissue. When an individual is homozygous for the defective (mutation) gene, wither no phenylalanine dehydroxylase or an insignificant amount of it is produced, as a result excessive concentrations of endogenous phenylalanine accumulates in the blood. This high level

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11of phenylalanine cause the myelin sheath around neuron axons of the central nervous system to be abnormally formed which leads to mental retardation. This inborn error have the frequency of 1 in 10,000 Caucasian newborns.

Adenosine deaminase deficiency (SCID)

Adenosine deaminase deficiency causes buildup of adenosine and deoxyadenosine levels in the circulatory system. These compounds are toxic and lethal to both B and T lymphocytes and as a consequence cause severe immunodeficiency.

Familial Hypercholesterolemia

The patient is homozygous recessive for familial hypercholesterolemia. This individual has no low-density lipoprotein (LDL) receptors on liver cells. As a result, fat bodies containing LDL cholesterol are not processes by the liver and continually circulate in the blood stream, leading to arterial blockage and severe heart disease. Such patients do not respond to drug treatment and benefits of coronary bypass surgery are short-lived. Treatment with ex vivo gene therapy may give an better option for a better life to these patients.

Methods of Introducing Exogenous DNA Into Mammalian Cells

1. Nuclear transformation2. DNA/ Calcium phosphate co-precipitate method3. Microinjection of eggs4. Electroporation 5. Lipofection 6. Use of viral vectors

Two Steps of Transformation

1. Transient transformation: • introduction of DNA into the cell (transfection stage)• DNA maintained in the nucleus as an extra-chromosomal state• If they lack replication origin then they persist for a short time before they

degrade• Application: short term experiment, rapid production of R-protein

2. Stable transformation:

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12• incorporation into the genome (integration stage)• small portion of the transfected DNA will incorporate• a new genetic locus will be inherited by all clonal progeny• application: long term analytical experiments and production of large amount of

r-proteinUse of Viral Vector for Transfection

Principal of virus used in transfection • Transfer DNA into cells• High level replication• Gene expression

Transfection through Viral vector is used to1. Gene expression in the cultured cells2. Gene transfer to animalse.g. Adenovirus, retrovirus, herpesvirus and adeno-associated virus and baculovirus.

Infection Cycle of Retrovirus in Host Cell

[Fig. The retrovirus life cycle (courtesy of Crucell). Following adhesion, the virion enters a cell and its RNA is reverse transcribed into a DNA molecule which is subsequently integrated into the cellular chromosome to form the provirus. Proviral DNA is transcribed and translated into viral proteins which encapsidate full length viral RNA molecules. The encapsidated viral RNA buds from the cell to give progeny virus particles.]

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13The gag gene codes for the core proteins of the virus that are responsible for the encapsulation of viral genomic RNA and the assembly of the virion. The pol gene encodes reverse transcriptase that directs the synthesis of the DNA from the viral RNA. The env (envelope) proteins are located at the surface of the virions and interact with specific cell surface receptors on the target cell membrane. Both proviral termini consist of a non-coding sequence, called the Long Terminal Repeat (LTR). The LTR contains promoter sequences responsible for making RNA from the provirus and for integration of proviral DNA into the chromosomal DNA of the host. Downstream of the LTR, a stretch of sequences of approximately 300 bp is present which is required for the packaging of the RNA genome into virions (so-called packaging sequence Ψ)

Strategy to Use Retrovirus Vector for gene transfer

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[Fig: A retrovirus packaging cell at work (courtesy of Crucell). The packaging cell by itself produces all of the viral proteins, but generates no viable virus particles. A retroviral shuttle vector carrying all of the in-cis requirements as well as the gene(s) of interest is introduced into the packaging cell, e.g. by physical transfection procedures. Transcripts from the shuttle vector can be packaged by the virus proteins present and form infectious virus. Virus stocks from such cells are therefore helper free and the recombinant retroviruses are replication defective such that they can only undergo one cycle of infection.]

14Recombinant retrovirus production systems is consists of two building blocks, namely a retroviral vector and a retrovirus packaging cell. Packaging cells are special cells. They contain the retroviral genes gag, pol and env and hence these cells by themselves synthesize all retroviral proteins (Valerio, 1992). However, the packaging cells themselves do not contain the LTR and the packaging sequence. Thus, packaging cells only make retroviral proteins gag, pol and env but are incapable of making retroviral particles.

Retroviral vectors are retroviral particles that have a retroviral RNA genome that contains, like wild-type retroviruses, the packaging signal and at each end a long terminal repeat (LTR). However, the retroviral genes gag, pol and env are replaced by a gene encoding a therapeutic protein "target cell”

When a retroviral vector infects a packaging cell (Figure), a provirus DNA is made which integrates into the host cell genome. The proviral DNA of the retroviral vectors does not contain the gag, pol and env genes. It contains the gene encoding the therapeutic protein. A packaging cell does make the gag, pol and env (the respective genes are present in the genome of the packaging cell, but not in the provirus) and the retroviral vector RNA is made and packaged into new particles similar to wild-type viruses. The newly generated virus particles are released into the culture medium.

Non-Viral Gene Delivery Systems

(In vivo Gene Therapy)

Advantages:1. Large DNA molecule can be transfected.

Disadvantages:1. The frequency of transfection is often too low to create a therapeutic effect.2. The duration of gene expression is short lived.

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15Non-Viral Gene Delivery Systems (Methods)

A. Gene Gun MethodB. LiposomeC. DNA-mediated conjugation delivery system

A. Gene Gun Method

Method: Gold particles (1 to 3 um in diameter) coated with pure DNA are directly propelled into tissue.Advantages:

1. Least complicated method.2. The gene is directly delivered into the target tissue. Propelled into skin or

released into the subcutaneous tumor cells through gene gun.Disadvantages:

1. Applicable to accessible tissues2. Require large amount of DNA3. Recombinant/ heterologous protein that are not part of the circulatory system

get inactivated or degraded.B. Liposome/ Lipoplex

Liposome: are Lipid share where DNA construct surrounded with lipid layer with an aqueous core facilities that passage of the DNA through the cell membrane.Lipoplex: This is a type of liposome. These are cationic liposome have a positive charge on the outside and bind to negatively charged DNA molecules to form a lipid-DNA complex.Advantages:

1. Nontoxic2. Non-immunogenic

Disadvantages:1. Gene transfer efficiency is not high.2. After cellular internalization large amount of DNA is taken up by liposome and

degraded.3. Expression duration is short.

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16C. DNA-Molecular Conjugate Delivery System

Method: First poly-L-lysine is chemically linked (conjugated) to a molecule that binds to a specific cell receptor. Then, DNA is added to this Poly-L-lysine linked molecule. Finally, DNA combines to form tightly compacted, twisted, solid ring (toroid). The cell receptor binding sites are arrayed on the surface of the DNA-Molecular conjugate.Advantage:

1. Large DNA molecules (>10 kb) can be transferredDisadvantages:

1. Transformation efficiency is low.

POLYMERASE CHAIN REACTION (PCR)

The polymerase chain reaction (PCR) provides a simple and ingeneous method for exponentially amplification of specific DNA sequences by in vitro DNA synthesis. This technique was developed by Katy Mullis at Cetus Corporation in Emery Ville, California in 1985. Katy Mullis shared the Nobel prize for chemistry in 1993. This technique has made it possible to synthesize large quantities of a DNA fragment without its cloning. It is ideally suited where the quantity of biological specimen available is very low such as a single fragment of hair or a tiny blood stain left at the site of a crime. The details of PCR techniques and its mechanism are described by Erlich (1989) in his edited book 'PCR Technology'. The PCR technique has now been automated and is carried out by a specially designed machine.

The PCR requires the following:

(i) DNA Template: Any source that contains one or more target DNA molecules to be amplified can be taken as template.

(ii) Primers: A pair of oligonucleotides of about 180-30 nucleotides with similar G+C contents act as primers. They direct DNA synthesis towards one another. The primers are designed to anneal on opposite strands of target sequence so that they will be extended towards each other by addition of nucleotides to their 3' ends.

(iii) Enzyme: The most common enzyme used in PCR is a thermo stable enzyme called Taq polymerase. It is isolated from a thermostable bacterium called Thermus aquaticus. It survives at 95°C for 1-2 minutes and has a half life for more than 2 hours at this temperature. The other thermostable polymerases can also be used in PCR.

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17Mechanism of PCR

STEP 1 : Denaturation (Melting of Target DNA)STEP 2 : Primer AnnealingSTEP 3 : Primer Extension (Polymerization)

Application of PCR

After the discovery of PCR, the modern biology has been revolutionized in each and every aspect. Some of the areas of application of PCR have briefly been discussed herewith.

01. Diagnosis of Pathogens: There are several pathogens that grow slowly. Therefore, their cells are found less in number in the infected cells/tissues. It is difficult to culture them on artificial medium. Hence, for their diagnosis, PCR-based assays have been developed. These detect the presence of certain specific sequences of the pathogens present in the infected cells/tissues. Besides, it is useful in detection of viral infection before they cause symptoms or serious diseases.

02. Diagnosis of Specific Mutation: In humans there are thousands of genetic diseases. Mutations are also related to genetic diseases. Presence of a faulty DNA sequence can be detected before establishment of disease. By using PCR sickle cell anaemia, phenylketonuria and muscular distrophy can also be detected. The other diseases can also be diagnosed by using PCR. For example PCRbased diagnostic tests for AIDS, Chlamydia, tuberculosis, hepatitis, human pappiloma virus, and other infectious agents and diseases are being developed. The tests are rapid, sensitive and specific.

03. In Prenatal Diagnosis: It is useful in prenatal diagnosis of several genetic diseases. If the genetic diseases are not curable, it is recommended to go for abortion.

04. DNA Fingerprinting: In recent years DNA fingerprinting is more successfully used in forensic science to search out criminals, rapists, solving disputed parentage and uniting the lost parentage uniting children to their parents or relatives by confirming their identity. This is done through making link between the DNA recovered from samples of blood, semen, hairs, etc. at the spot of crime and the DNA of suspected individuals or between child and his/her parents/relatives.

05. In Research: In addition, DNA fingerprinting of new microorganisms isolated from various extreme environment (soil, water, sediments, air, extreme habitats, etc.)

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18is also carried out to confirm their identity by comparing with the DNA sequences of known microorganisms. Their DNA and RNA can be amplified. Besides, it is also useful to determine the orientation and location or restriction fragments relative to one another.

06. In Molecular Archaeology (Palaentology): PCR has been used to clone the DNA fragments from the mummified remains of humans and extinct animals such as the woolly mammoth and dinosaurs from the remains of ancient animals as recently epitomized in Michel Crighton's Jurassic Park. DNA from buried humans has been amplified and used to trace the human migration that occurred in ancient time.

07. Diagnosis of Plant Pathogens: It is also applied in diagnosis of plant diseases. A large number of plant pathogens in various hosts or environmental samples are detected by using PCR, for example, viroids (associated with hops, apple, pear, grape, citrus, etc), viruses (such as TMV, cauliflower mosaic virus, bean yellow mosaic-virus, plum pox virus, potyviruses), mycoplasmas bacteria (Agrobacterium tumifticiens, P,Yeudotiioticis,vol(in(iceartini, Rhizobium legunfinosaritin, Xanthol"onas compestris, etc), fungi (e.g., collectotrichum gloeosporioides, Glomus spp., Laccaria spp., Phvtophthora spp., Verticillium spp), and nematodes (e.g. Meloido,,,,vne incoginta, M. javanica, etc) (Henson and French, 1993; Chawla, 1998).

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