Cloning is the creation of an organism that is an exact genetic copy of another. This means that every single bit of DNA is the same between the two! Cloning is done by putting a foreign piece of DNA into a usually circular DNA that acts as a carrier. This circular DNA is called a vector, which is a means of transport. The veCloning is the production of one or more individual plants or animals that are genetically identical to another plant or animal. Nature itself is the greatest cloning agent. In about one of every 75 human conceptions, the fertilized ovum splits for some unknown reason and produces monozygotic (identical) twins. Each has an genetic makeup identical to the other.
A carrier such as bacterial plasmid or bacteriophage used to insert a genetic sequence such as deoxyribonucleic acid fragment or complete gene , into a host cell such that the foreign genetic material is capable of replication. DNA from
Cloning in biology is the process of producing populations of genetically-identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms.
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with the same restriction enzyme, then ligating the fragments together. There are many types of cloning vectors. Genetically engineered plasmids and bacteriophages (such as phage λ) are perhaps most commonly used for this purpose.
Cloning vectors for animal
Cloning in animals can be accomplished by inserting the DNA of one animal into the foetus of the same specie along with a vector which gets cleans out the DNA already in the embryo. Sometimes the vector does not get rid of the original DNA of the foetus or the foetus does not adopt the new DNA.
Considerable effort has been put into the development of vector system for cloning genes in animal cells. These vectors are needed in biotechnology for the synthesis of recombinant protein from genes that are not expressed correctly when cloned in E. Coli or yeast , and methods for cloning in humans are being sought by clinical molecular biologist attempting to devise techniques for gene therapy, in which a disease is treated by introduction of a cloned gene into the patient.
The clinical aspect has meant that most attention has been directed at cloning system for mammals, but important progress has been made with insects. Cloning in insects is interesting because it makes use of a novel type of vector that we have not met so for. We will therefore examine insect vectors before concluding the chapter with an overview of the cloning methods used with mammals.
Cloning vectors for insects
The fruit fly, Drosophila melanogaster, has been and still is one of the most important model organism used by biologists. Its potential was first recognised by the famous geneticist Thomas Hunt Morgan, who in 1910 started to carry out genetic crosses between fruit flies with different eye colours, body shapes and other inherited characteristics. These experiments led to the techniques still used today for gene mapping in insects and other animals. More recently, the discovery that the homeotic selector genes of Drosophila- the genes that control the overall body plan of the fly – are closely related to equivalent genes in mammals, had led to Drosophila melanogaster being used as a model for the study human developmental processes. The important of the fruit fly in modern biology makes it imperative that vectors for cloning genes in this organism are available.
P element Vectors
The development of cloning vectors for Drosophila has taken a different route to that the followed with becteria, yeast, plants and mammals. No plasmids are known in Drosophila and although fruit flies are like all organisms, susceptible to infection with viruses, these have not been used as the basis for cloning vectors. Instead, cloning in Drosophila makes use of a transposon called the P elements.
Transposons are common in all types of organisms .they are short pieces of DNA (usually less than 10kb in length) that can move from one position to another in chromosomes of cell. P elements which are one of several types in transposon in Drosophila are 2.9kb in length and contain three genes flanked by short inverted report sequences at either end of the element. The gene code for transposase, the enzyme that carries out the transposition process and the inverted repeats form the recognition sequences that enable the enzyme to identify the two ends of the inserted transposon.
As well as moving from one side to another within a single chromosome, p elements can also jump between chromosomes, or between a plasmid carrying a p element and one of the fly’s chromosomes. The latter is the key to use of P elements, one of which contains the insertion site for the DNA that will be cloned. Insertion of the new DNA into this P elements results in disruption of its transposase gene, so this element is inactive. The second P element carried by plasmid is therefore one that has intact version of the transposase gene. Ideally this second element should not itself be transferred to the Drosophila chromosomes, so it has its wings clipped: its inverted repeats are removed so that the transposase does not recognize it as being a real P element. Once the gene to be cloned has been inserted into a vector, the plasmid DNA is microinjected into fruit fly embryos. The transposase from the wings-clipped P element directs transfer of the engineered P element into one of the fruit fly chromosomes. If this happens within a germline nucleus then the adult fly that develops from the embryos will carry copies of the cloned gene in all its cells. P element cloning was first developed in the 1980s and has made a number of important contributions to Drosophila genetics.
Baculovirus has played an important role in gene cloning with other insects. These vectors have been developed for transfection of insects. Two nuclearpolyhedrosis viruses (NPV), e.g., AcNPV (Autographa californica NPV) and BmNPV (Bombyx mori NPV), has been exploited for this purpose. The NPV polyhedrin has a very strong promoter, and the polyhedrin protein is not needed for NPV replication. Therefore, the general strategy is to replace the NPV polyhedrin coding sequence by the DNA insert so that the polyhedrin promoter drives the expression of the transgene. The recombinant NPV vectors from virions, infect silk worm larvae or cultured, and replicate to yield upto 50 microgram vector DNA per larva. BmNPV vectors are used for infection of silkwarm larvae while AcNPV vectors are multiplied and expressed in the larvae or cultured cells of the insect Spodoptera frugiperda.
Most of the animal vectors are designed to replicate and express in animal cells; only the passive transducing SV40 vectors are incapable of replication. Retrovirus and transposon vectors integrate into the genomes of host cells in a manner similar to the natural retroviruses and transposon, respectively. Both circular and linearized vectors are integrate into the host genome, but the latter (linearized vectors) are for more readily integrated than the former. It has also been found that presence of additional vector DNA along with the integrated than the gene construct interferes with the expression of introduced gene or transgene. Therefore it is often desirable to introduce the transgene with a minimum of vector DNA associated with it.
A Fish Vector:-
A typical fish vector is a plasmid, e.g. , pRSV. These vectors usually contain a selectable marker, e.g. , ampicillin resistance, and the origin of replication from E.coli plasmid pBR322, and an enhancer/promoter sequence (SV40 or roux sarcoma virus promoter), a multiple cloning site for insertion of the DNA insert and a termination site including the polyadenylation site . In addition , it has the SV40 origin of replication as well.
MAMMALIAM VIRUS VECTORS
The first animal vector was devised from the primate papova virus , simian virus 40(SV$); it was used for cloning in 1979. But subsequently , vectors have been developed from many other virus,e.g., papilloma virus, adenoviruses,the Epstein-Barr herpes virus, vaccinia viruses(all for mammals), and baculoviruses (for inserts).Recently during 1990s, mammalian artificial chromosomes (MACs) and human artificial chromosomes (HACs) have been developed.
SV40 is a spherical virus with a circular, double stranded 5,243 bp chromosomes, which incodes 5 proteins, viz., small-t, large-t (both early protein), VP1, VP2 and VP3 (VP= virion protein),has an origin of replication(about 80 bp) and is complexed with histones to form chromatin. Large-T is essential for viral replication, while VP1, VP2 and VP3 form the viral capsid. In laboratory, it is multiplied in cultured kidney cells of African green monkey; infected cells lyse after 4 days releasing upto 10 raise to the power 5 virions/cell. SV40 genome has been used to develop mainly the following three types of vectors: (1) transducing vectors (2) plasmid vectors and (3) transfomiong vectors.
These vectors produce viral particles after infecting monkey cells. They must have the following 3 features
1. SV40 origin including the surrounding region containing the transcriptional regulatoy signals.(i.e. regions at which splicing and polyadenylatin occurs.)
2. Total size(including that of the DNA insert)between 3900 bp and 5300 bp for packing into virions and
3. Genes encoding large –T, VP1,VP2 and VP3
This leaves very little room for DNA inserts. But the genes encoding the necessary proteins, wiz.,large-T,VP1,VP2 and VP3 can be present in other virus or within host genome. This flexibility is to advantage for solving the size and selection problems.
Late Replacement Vectors :
The recent encoding VP1,VP2 and VP3 may be replaced in the vector by DNA insert; such a vector is called late-region replacement vector, e.g- SVGT-5(an improved SV40 vector). A vector of this type contains the following :
1. The origin of replication.
2. The region at which splicing and polyadenylation occurs.
3. The entire early region of SV40 genome
Such a vector is used for the infection of host cells in conjunction with another virus called helper virus which has the VP1,VP2 and VP3 genes intact what has a defective large-T gene(a gene in the early region)gene. The large-T function is intact in and provided by the late replacement vector. Therefore , in this case only those host cells that are infected by both the vector and the helper virus will lies and produce virions since cells infected by either the vector or the helper virus alone will not support packing or replication (respectively) of the virus. This feature is very useful since all the plaques formed on monkey cells monolayers contain the vector.
Early Replacement Vector :
Alternatively the essential genes missing from the vector may be present within the genome of the host cells e.g. , COS(CV-1,origin of SV40; CV1 is a monkey cell line)cell line of African green monkey kidney cell cultures contains in is genome the gene for large-T of SV40. Therefore a vector having the origin o replication and genes for VP1,VP2 and VP3 will replicate and will produce virions in COS cell line cells. In such a case no helper virus is required. Since in such a vector the early genes(large-T gene) are replaced by the DNA insert, it is called early-region replacement vector .
The chief advantages of both late-an early-region the replacement SV40 vectors are as follows:
1. The recombinant DNA produces virions, which introduce the DNA into host cells by infecting them like SV40 virions.
2. The recombinant DNA replicates to a high copy number which is a distinct advantage.
These vectors have the following 2 limitations:
1. The expressions of transgenas in transient in the infector cells.
2. The maximum size of DNA insert is approximately 2.5 kb.
SV40 Plasmid Vectors:
These vectors replicate in monkey cells but don’t get packaged into virions. They contain the origin of replication and the large-T encoding gene(large-T gene is not necessary for multiplication in COS cells). Obviously, there is no size limit on such vectors . and some of them are E.coli and the monkey shuttle vector, e.g.pSV2,pSV3,etc. These vectors produce high copy number per COS cell; this makes the host cells inviable. Therefore , permanent COS cell lines having recombinant DNA are not obtained, but transient expression of cloed genes can be analysed . The shuttle vectors are used to propagate the recombinant DNA in E.coli, which are then introduced into monkey cells to study the expression of DNA inserts . Plasmid vectors can be constructed by using the origin and the early region of polyma virus in the place of those of SV40.
pSV40 (plasmid simian virus)vectors are constructed by incorporating the SV40 sequences into pBR322. The bacterial XGPRT gene integrated after the SV40 early promoter sequence serves as selectable marker when the vector is transferred into mammalian cells . pRSV series of vectors are derivatives of pSV vectors. Plasmid vectors are unstable in monkey cells, and are generally used for transient transfection only. These vectors can be stably maintained if the large-T function is provided by COS cells and the vector has a selectable marker and the host cells are maintained under selection environment . Plasmid vector also integrate in the host cells genome with the frequency of 10 raise to the power of -5 to 10 raise to the power of -3; this yields stable tansfection.
Non Replacing Vectors:
These vectors don’t replicate . They only serve as vehicles for transfecting DNA’s, which may become integrated into the host DNA; such vectors are ,therefore , also called passive tansfecting vectors. These vectors are generally shuttle vectors: they are first cloned in E.coli to isolate recombinant DNA and then used to transfect various mammalian cells since they need not be restricted to monkey cells, in view of their lack of replication. The SV40 sements used in these vectors are generally the transcription regulatory sequences and the polyadenylation sites. Permanent transfectants are selected on the basis of selectable markers present in the vector.
The selectable marker, need not be covalently link to the DNA insert o the transfecting DNA. Even when 2 separate DNA fragments containing separate genes are mixed and used for infection , more than 50% of the permanently transfector cells contain both the genes, usually integrated side by side. The 2 DNA fragments tend to become joined after entering the animal cells, which is the reason for their co-transformation, i.e. integration of 2 genes together in the genome.
Papillomavirus belongs to papovavirus class, and cause warts are approximately 7.9 kb genome organised in nucleosomes. The bovine papillomavirus (BPV) replicates as a stable plasmid in rodent and bovine cells and the cells are not killed; the virus behaves as a multicopy(upto 100 copies per cell) and is passed to daughter cells on cell division . The viral genome transforms cells, which behave like tumour cells and form piled up colonies of cells ionstead of the typical monolayer. The tansformed state is due to the genes present in the ‘transforming region’ (about 5500 bp)of the virus genome. The virus genome is generally used to produce shuttle vectors by using the transforming region of the genome.
Retoviruses have single-stranded RNA genome. Each virus has 2 copies of genome, which resemble eukaryotic mRNAs . The viral genome reverse transcribed by reverse transcriptase into a DNA double strand copy inside the host cells. The DNA copy integrates into the host genome to become a provirus, which cause permanent transfection of the cells. The povirus genome is transcribed and expressed ; virions are formed and extruded into the medium. The following properties of the retroviruses are useful in their use as vectors:
1. A wide host range (Birds ,mammals and other animals)
2. Infected cells are not killed and they continue to produce virus particles over indefinite period.
3. Presence of strong promoters.
4. Regulation of promoter action in case of some viruses viz , murine mammary tumour virus.
Retroviral vectors are constructed from cloned DNA genomes of retroviruses. These have the following 5 features:
1. The vector has the viral sequences for replication, gene expression and packaging
2. DNA inserts may either replace or be located in the non essential coding regions of the viral genome.
3. The vector and the recombinant DNA are packaged into the virions and used as transducing viruses.
4. The viral proteins are usually provided by the helper virus or a provirus.
5. DNA copies of the retovirus genome are used as vectors ,generally as shuttle vectors.
Vaccinia Virus Vectors:
Vaccinia virus genome has been used to construct vectorsfor cloning of genes of pathogen region and of other such genes that facilitate the use of recombinant vaccinia virus as live vaccineas.
Adenoviuses are DNA viruses; their genome of double –stranded DNA of approximately 36kb . Reconbinant viruses , DNA insert replaces genes EIA/EIB;these genes regulate transcription and are essential for virus replication. Theefore , recombinant viruses are propagated in a transfacted cell line that constitutively express EIA/EIB genes. Gene E3 o adenoviruses is usually deleted in addition to EIA/EIB.
MAMMALION ARTIFICIAL CHROMOSOMES (MAC) VECTORS
Mammalian artificial chromosomes and human artificial chromosomes vectors are linear vectors that contain centromeres , telomeres , origin of replication recognition sequences
And transcriptionally active chromrsomal domains. These vectors esemble microchromosomes , are mitotically and cytogenetically stable in the absence of any selection, bind centromere proteins specific for active centomeres,viz CENP-B,CENP-C and CENP-E and are about 6-10 megabases in size. MAC’s and HACs can be contucted in cultured cells by artificially changing the structure of natural chromosomes. Alternatively, chromed chromosomal elements may be transferred into cultured cell leading into the de novo formation of MACs and HAcs.
The chromosomel sequences required for the de novo construction of MACs and HACs are so large that their cloning in a single molecule is extremely difficult. Instability during cloning of repetitive telomeric and centromeric DNA further complicates MAC/HAC construction. In addition, such large DNA molecules are not stable in solution; therefore their isolation, manipulation and transfer are very problematic . MACs and HACs are expected to provide a strategy for an efficient and predictable production of transgenic animals, and to a safe and efficient somatic gene therapy in humans.