Manual Rabbit Biotechnology: Rabbit genomics, transgenesis, cloning and models

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Because of its larger size, surgery manipulation, bleeding, and turn-over studies are much easier performed in rabbits than in mice.

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Furthermore, transgenic rabbits can be produced using microinjection and other methods such as lentiviral v- tors. Cloning in rabbits has been proved possible, even though still laborious and time-consuming. Hopefully, functional rabbit ES cell lines will be available in the coming years. Gene deletion or knock-out in rabbits will then become possible. Rabbits as laboratory animals. Sciences de la vie. Animals, Genetically Modified. Genetic Engineering. Bibliographic information.

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ISBN Librarian view Catkey: The type of host cells used in a particular application will depend mainly on the purpose of the cloning procedure. Host cells exploited are modified bacterial, fungal cells e. Yeast , or still virus, being the bacterial system e. The vector may have an origin of replication that originates from either a natural extrachromosomal replicon or, in some cases, a chromosomal replicon [ 11 ]. Besides the structure the vectors should contain a sequence that make possible to select the recombinant cells, like a marker gene and in third place they should contain restriction sites into which the DNA can be inserted [ 29 ].

The types of cloning vectors are plasmids, phages, cosmids, phagemids, artificial chromosomes, viral vector and transposons. Each of them will be briefly describe in this section. Plasmids are small circular double-stranded DNA molecules, which exist in the cell as extrachromosomal units. In a cell, they have the ability for self-replicating, and copy numbers maintenance.

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Due to their capacity of copy numbers they can be classified as: single copy plasmids or multicopy plasmids. The single copy plasmids are maintained as one plasmid DNA per cell, instead the multicopy plasmids that are maintained as copies per cell. Another kind of plasmids consists in ones that are under relaxed replication control, allowing their accumulation in numbers up to copies per cell, being the used ones as cloning vectors [ 15 ]. The plasmids vectors are designed to work in bacteria cells.

An important property in these vectors is the detection of the same in the host cells. Usually, the detection mechanisms are done through antibiotic resistance. The host cell strain chosen is sensitive to a particular antibiotic and the plasmid is designed to contain a gene conferring resistance to this antibiotic. According to P. Gupta [ 15 ], there were three phases of plasmid development cloning vectors. The first included the plasmids pSC, ColE1 and pCR1, which are naturally occurring plasmids, and not suitable for efficient cloning, since plasmid can transfer the gene through bacterial conjugation or can be integrated in the bacterial genome having no accessible detection system.

Other disadvantage lies on having no more than two restriction sites for cloning. The drawbacks of naturally occurring plasmids were overlapped by pBR and pBR The size reduction brought the pBR, which was largely used for many years. The second phase relies on reducing the plasmids sizes, because the transformation efficiency and vector size have a proportional inverse relation.

This plasmid vectors incorporate the selection mechanism of antibiotic resistance described above. Nowadays, there are a lot of plasmids commercially available that can be purchased depending on the application needs.

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A bacteriophage lambda is a bacterial virus that infects E. Its utility as a cloning vector depends on the fact that not all of the lambda genome is essential for its function [ 1 ]. The lambda genome has the left-hand region with essential genes for the structural proteins and the right-hand region has genes for replication and lysis, while the middle region has the genes for integration and recombination, which are non-essentials.

There are two possible types of lambda vectors: the insertion vector and the replacement vector. The insertion vector has only a single recognition site for one or more restriction enzymes, enabling the DNA fragment to be inserted into the lambda genome. The lambda particle integrates DNA molecules between 37 and 52kb, and to adapt longer inserts is necessary to remove some of lambda genome.

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  • The region for replacement is the middle one where, more 23 kb of foreign DNA can be inserted. This vector is known as replacement vector [ 28 ]. The replacement vector cannot be integrated into the host cells chromosome being necessary to use a helper phage to provide integration and recombination functions.

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    On the other hand, this vector has two restriction sites, having a whole section of phage genome being replaced during cloning [ 1 ]. M13 is filamentous bacteriophages that infect specific E. Your attractive as a cloning vector consists in its genomes contain the desirable size for a potential vector less than 10kb ; does not kill the host when progeny virus particles are released and thus, is easily prepared from an infected E.

    Besides, M13 is used as cloning vector to make single stranded DNA for sequencing and mutagenesis approaches. The M13 genome is a single-stranded DNA molecule with bp in length. This bacteriophage only infects bacteria carrying the F-pili fragile protein appendages found on conjugation-proficient cells , being male-specific. When the DNA enters the cell, it is converted to a double-stranded molecule known as replicative form, which is a template for making about copies of the genome. At this point replication becomes asymmetric, and single-stranded copies of the genome are produced and extruded as M13 particles.

    The property of do not lyse the host cell brings a DNA resource, although growth and division are slower than in non-infected cells [ 1 , 11 , 28 ]. Cosmids are plasmid particles into which certain specific DNA sequences, namely those for cos sites, are inserted. The goal of these vectors development is to cloning of large DNA fragments up to 47kb in length. The advantages consist of a highly efficient method of introducing the recombinant DNA and, a cloning capacity twofold greater than the best lambda replacement vectors.

    On the other hand, the gains of using cosmids instead of phage vectors are offset by losses in terms of ease to use and further processing of cloned sequences [ 1 ]. The methodology to use the cosmid cloning vectors consists in put together the cleaved vector and the target DNA for cloning, producing concatameric molecules. The concatameric molecules are usually generated by first linearizing the cosmid so that each end has cos site. Partial digestion leaves some site uncut and allows large segments of a genome to be isolated.

    These segments are mixed with the two halves of cosmid and joined using ligase. Thus, these molecules are packaged into phage heads by mixing with a packaging extract, becoming infectious.

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    Phagemids combine desirable features of both plasmids and bacteriophages. The construct consists of a plasmid with a segment of a filamentous bacteriophage, such as M13, having two different origins of replication: the plasmid and the phage origin. The selected phage sequences contain all the cis -acting elements required for DNA replication and assembly into phage particles [ 11 , 30 ]. These vectors allow successful cloning of inserts several kilobases. After E. These particles contain a mix of recombinant phagemids and helper phage.

    Vector pairs that have the phage origin in opposite directions are available, and as a result single stranded DNA representing of both DNA strands are produced. This mixed single strand DNA population can be used directly for DNA sequencing, if the primer for initiating DNA synthesis is designed to bind specifically to sequences of phagemid adjacent to the cloning site [ 11 , 30 ]. A bacterial artificial chromosome BAC is a single copy bacterial vector based on a functional fertility plasmid F-plasmid of E. BAC vectors are superior to other bacterial system, due to the F factor, which has genes regulating its own replication and controlling its copy number.

    These regulatory genes are oriS and repE , mediating unidirectional replication and parA and parB , maintaining the copy number to one or two per cell.

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    The cloning site is flanked by T7 and SP6 promoters for generating RNA probes for chromosome walking and for DNA sequencing of the inserted segment at the vector-insert junction. The CosN and loxP sites provides convenient generation of ends that can be used for restriction-site mapping to arrange the clones in an ordered way [ 31 ].

    Besides the maintenance of large DNA inserts, BAC has structural stability in the host, high cloning efficiency and easy manipulation of cloned DNA, being largely utilized for construction of DNA libraries from complex genomes and subsequent rapid analysis of complex genome structure [ 31 ].

    Transformed suitable E. The selection of recombined cells is done by hybridization procedures. Viral vectors are commonly used to deliver genetic material into cells for gene therapy due to specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. This process can be performed inside a living organism in vivo or in cell culture in vitro , being frequently used to increase the frequency of cells expressing the transduced gene [ 32 ]. The first use of vector virus for cloning was based on simian virus 40 SV40 , a polyomavirus originated of rhesus macaque, being a potent DNA tumor virus infecting many types of mammal cells in culture.

    The SV40 genome is 5. Due to packing limitations, cloning with SV40 involves replacing the existing genes with the foreign DNA [ 32 - 34 ]. Adenoviruses came to solve the size of insert drawback of SV40, enabling the cloning of DNA fragments up to 8kb. On the other hand, due to its larger genome, adenoviruses are difficult to handle. Expression can be transient and the in vivo transfection can be impaired due to immune response. Papillomaviruses also have a high capacity for inserted DNA with the advantage of stable transformed cell line.