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J.Bio.Innov2(5),pp:240-259,2013 www.jbino.com
College of Veterinary Science and Animal Husbandry, Rewa-486 001 (M.P.), INDIA
Email: niteshprof@gmail.com
(Received on date: 25th August 2013 Date of Acceptance: 20th September 2013)
ABSTRACT
The emergence of transgenic technology has widened the scope of development in case of farm
animals and the advent of new molecular biology techniques has paved way by giving a new
dimension to animal breeding. The transgenic technology is one of an important tool to meet the
future challenges for increased animal’s production. The biological products from animal source
should be handled with safety as they are subject to contamination and could be damaged very
easily. Thus, safety guidelines should be developed for the commercial exploitation of
recombinant proteins and ensure that the transmission of pathogens from animals to human
beings is prevented. Therefore, the genetically engineered animals and biotechnology will play a
vital role in the production of pharmaceutical proteins and bring about a complete refinement in
agriculture production by increasing the quality and quantity of production, protection of
environment, maintenance of genetic diversity and overall improvement in animals welfare.
J.Bio.Innov2(5),pp:240-259,2013 www.jbino.com
J.Bio.Innov2(5),pp:240-259,2013 www.jbino.com
Ruddle, 1981) and then to various other research are mice. Over 80% of mouse
species such as rats, rabbits, sheep, pigs, genes function the same as those in humans.
birds, and fish. Two other main techniques Mice also have a short reproduction cycle
were Subsiquently developed: those of and their embryos are amenable to
retrovirus-mediated transgenesis (Jaenisch, manipulation. Mice are therefore an ideal
1976) and embryonic stem (ES) cell- human surrogate in the study of most
mediated gene transfer (Gossler et al., diseases. It is hoped that the refinement of
1986). transgenesis techniques in mice will
After 1981, when the term transgenic was ultimately allow for a corresponding
first used by J.W. Gordon and F.H. Ruddle reduction in the use of "higher" animals,
(1981), there has been rapid development in such as dogs and non-human primates, in
the use of genetically engineered animals as biomedical research. Other transgenic
investigators have found an increasing animals include rats, pigs and sheep. An
number of applications for the technology. example:Normal mice cannot be infected
with polio virus. They lack the cell-surface
What is Transgenic Animal? molecule that, in humans, serves as the
A transgenic animal is one that carries a receptor for the virus. So, normal mice
foreign gene that has been deliberately cannot serve as an inexpensive, easily-
inserted into its genome .It is the one which manipulated model for studying the disease.
has been genetically altered to have specific However, transgenic mice expressing the
characteristics it otherwise would not have. human gene for the polio virus receptor can
In animals, transgenesis either means be infected by polio virus and even develop
transferring DNA into the animal or altering paralysis and other pathological changes
DNA of the animal. Transgenic animal are characteristic of the disease in humans.
genetically modified to contain a gene from
a different species following gene Why Transgenic Animal?
transplantation or resulting from the Interest in transgenic animals originally fall
molecular manipulations of endogenous into two broad categories:
genomic DNA. The new gene is inherited by To increase production efficiency of farm
offspring in the same way as the organism’s animals in a short duration.
own genes. The earliest transgenic Molecular farming: Using livestock to
approaches involved transferring DNA, produce medicines, nutraceuticals and
usually by injection into a fertilised mouse tissues for transplant into humans.
egg. However, since it is not possible to
control the site of integration of the foreign Strategies for Producing Transgenic
DNA using this technique, it is a relatively Animals
imprecise tool. Mice resulting from this There are two basic strategies for producing
technique are generally called transgenic animals, which include “gain of
"overexpressors". Currently over 95% of function” or “loss of function” transgenics.
transgenic animals used in biomedical The basic idea behind the gain of function
J.Bio.Innov2(5),pp:240-259,2013 www.jbino.com
strategy is that by adding a cloned fragment vectors, retrovirus vectors etc., behave like
of DNA into an animal’s genome to a new viruses in that they produce virions, which
gene product is produced that did not are used to infect the host cells. Some other
previously existed in that cell or tissue. E.g. vectors are like bacterial plasmids, e.g., SV
expression of human growth hormone 40 plasmid vectors, bovine papillomavirus
(hGH) in mouse liver and to get over vectors and polyoma virus vectors; these
expression of gene product in the proper vectors have to be introduced in the cell
tissue (Palmiter et al., 1982). using a suitable transfection technique.
The loss of function approach has many Drosophila P elements have been developed
similar applications as the gain of function as valuable vectors for this invaluable
strategy especially in view of over genetic material. The 31 bp inverted repeat
expression, insertional mutations and borders and the neighboring sequence of P
antisense situations. This strategy relies on elements are combined with a suitable E.coli
the ability of the embryonic cells to undergo plasmid, e.g., pUC8, to produce a shuttle
homologous recombination (“gene vector. DNA insert of up to 40 kb can be
targeting”).Gene targeting permits the placed between the two border sequences.
transfer of genetic alteration created invitro The recombinant P DNA is injected into
into precise site in the embryonic or cell Drosophila larvae along with a helper P
genome. If the host’s cells are totipotent or element, which produces transposase.
pluripotent embryonic cells or Transposase enables the transposition of
reprogrammable somatic cells, these recombinant P element (carrying the DNA
homologous recombination events can be insert) from the recombinant DNA into the
transferred to the germ line of the offspring. Drosophila genome.
This strategy has extraordinary potential for Bacculovirus vectors have been developed
making specific genetic changes for use in for transfection of insects. Two nuclear
medicine; agriculture and for further polyhedrosis viruses (NPV), e.g. AcPNV
understanding of the genetic control of (Autographa californica NPV) and BmNPV
developmental processes. (Bomby x mori NPV) have been exploited
for this purpose. The NPV polyhedron
Vectors protien gene has a very strong promoter, and
Vectors are plasmid or viral DNA employed the polyhedron protein is not needed for
in recombinant DNA technology to clone a NPV replication. Therefore, the general
foreign gene in prokaryotic or eukaryotic strategy is to replace the NPV polyhedron
cell. The various animal vectors are based coding sequence by the DNA insert so that
on one or the other virus e.g. SV40 vectors, the polyhedrin promoter drives the
bovine papillamovirus vectors, retrovirus transgene. The recombinant NPV DNA form
vectors, etc., or on a transposable element virions, infect silkworm larvae or cultured
e.g. Drosophila P element vector. It may be cells, and replicate to yield upto 50 ug
pointed out that some of the these vectors vector DNA per larva. BmNPV vectors are
e.g., early or late region replacement SV40 used for infection of silkworm larvae, while
J.Bio.Innov2(5),pp:240-259,2013 www.jbino.com
AcNPV vectors are multiplied and different types of vectors used for gene
expressed in the larvae or cultured cells of transfers in animals are shown in Table 1.
the insect Spodoptera frugiperda. The
Table 1: The different types of vectors used for gene transfers in animals
J.Bio.Innov2(5),pp:240-259,2013 www.jbino.com
It may be seen from table that most of the integrate into the host genome, but the latter
animal vectors are designed to replicate and are far more readily integrated than the
express in animal cells; only the passive former. It has also been found that the
transducing SV 40 vectors are incapable of present of additional vector DNA along with
replication. Retrovirus and transposon the integrated gene construct interferes with
vectors integrate into the genomes of host the expression of introduced gene or
cells in a manner similar to the natural transgene. Therefore, it is often desirable to
retroviruses and transposons, respectively. introduce the transgene with a minimum of
Both circular and linearized vectors can vector DNA associated with it.
J.Bio.Innov2(5),pp:240-259,2013 www.jbino.com
J.Bio.Innov2(5),pp:240-259,2013 www.jbino.com
Fig. 2: Show the embryonic stem cells in-vitro increases the efficacy of production of transgenic
animals
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J.Bio.Innov2(5),pp:240-259,2013 www.jbino.com
method allowing the targeted amplification and PCR techniques detect the transgene
of specific regions of DNA from a sample. irrespective of whether it is integrated in the
The method is rapid, giving diagnostic genome or is present in an
results in a matter of hours, and sensitive, extrachromosomal state. Therefore, dot blot
requiring very small amounts of starting or PCR assay positive individuals are
material (<1ng). subjected to Sothern hybridization, for
Transgenic individuals are most easily confirmation of transgene integration etc.
identified when the transgene produces a PCR has applications in:
distinct phenotypic effect, but such cases are Rapid screening (2-4 hrs) for
only occasional. A more general approach identification of transgenic animals.
utilizes either dot-blot technique of PCR Detection of small amounts of transgenes
amplification using genomic DNAs in samples of blood or other tissues.
extracted from tail biopsies from 6-7 weeks Detection of mosaicism within an
old mice. In case of fish, genomic DNA is individual.
usually extracted from pectoral fin tissue. Rapid assessment of environmental
PCR amplification can be used if the contamination / biosafety breaches.
transgene has unique sequences, not found Due to the ease of use, speed and
in the host genome that can be used as sensitivity, standard PCR remains an
primers. This allows very small amounts of important tool especially for identification
DNA from presumptive transgenic of animals possessing the transgene of
individuals to be suitably amplified for a interest.
reliable detection of the transgene. The PCR Real-time quantitative PCR (Q-PCR)
approach is briefly described below: The real-time PCR system is based
1. The test DNA is amplified using unique on the detection and quantification of a
transgene sequences as primers in a PCR. fluorescent reporter. The fluorescence
2. The amplified DNA is subjected to signal increases in direct proportion to the
agarose gel electrophoresis; the transgene amount of PCR product in a reaction (Heid
construct is also run as a control. et al., 1996). This system has enormous
3. DNA from the gel is blotted onto a solid application within the area of transgenics.
support following the protocol for Q-PCR has applications in:
Southern blotting. The same samples as for standard PCR.
4. A radioactive probe specific for the Automated, high-throughput screening of
transgene is used for hybridization, and samples.
the hybridizing samples are detected by Accurate quantification of the amount of
autoradiography in the same manner as transgenic DNA within a sample.
for Southern hybridization.
The samples that test positive for Q-PCR is likely to supersede standard PCR
hybridization with the probe are from for most applications, because it is
putative or suspected transgenic individuals. quantitative. Set-up costs of the technology
It should be kept in mind that both dot blot
may be prohibitively high for smaller
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J.Bio.Innov2(5),pp:240-259,2013 www.jbino.com
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Engelhardt, 2003;) Pigs could be used as an feed conversion (Nottle et al., 1999).
effective model for the study of growth
hormone releasing hormone (GHRH) Milk production and Lactation
defects (Draghia – Akli et al., 1999). The advances in transgenic technology
provide ample chances to improve both the
(B) Livestock Production quality and quantity of milk produced. The
There are many potential applications of animals could be made to secrete
transgenic technology in producing new nutraceuticals in milk that may have an
varieties of livestock that has increased impact over the growth of offspring. Casein
growth rate, reproductive performance, feed variants are the main target for improving
utilization, improved milk production and the milk composition, which in turn alters
high disease resistance. Other by products the physio-chemical properties of milk.
like meat and eggs also could be modified Brophy et al. (2003) reported that cloned
by this technology. transgenic cattle have been developed that
produce increased amounts of beta and
Carcass Composition and Growth kappa casein in milk that increase the value
Enhancement of milk in the production of milk based
Transgenic animals with exogenous gene products like cheese, yoghurt and also
constructs have been produced which has increase the shelf life of milk products.
enhanced growth rate and improved quality Transgenic animals also could be developed
of food. Growth hormone and insulin like to produce “infant milk” that has increased
growth factors genes have been expressed at levels of human lactoferrin, to generate
different levels in transgenic animals lactose free milk for lactose in tolerance
(Seamark, 1987). Transgenic cattle and populations by inhibiting the expression of
salmon fish have been produced that lactalbumin locus and to produce
contains foreign gene constructs. The hypoallergenic milk by knocking of down
introduction of chicken ski gene has caused the expression of B-lactoglobulin gene.
muscular hypertrophy in case of pigs (Pursel Transgenic animals could also be made to
et al., 1999) and cattle (Bowen et al., 1994). secrete antibodies in their milk that give
The acid meat gene or Rendement Napole resistance against several diseases like
gene has been involved in low processing mastitis or to secrete antimicrobial peptides
yields of pork there by affecting the quality like lysozyme. Grosvenor et al. (1993)
of meat in pig. Silencing the expression of reported that the milk composition could
this gene in case of pigs alter the post also be altered by making the transgenic
mortem pH and improve the quality of meat. animals to secrete growth factors in milk,
Other genes like GH releasing factor, IGF which in turn affect the growth and
binding proteins also play a major role in the maturation of newborn offspring.
modification of growth. Transgenic pig with
human metallothionein promoter had a
significant improvement in growth rate and
J.Bio.Innov2(5),pp:240-259,2013 www.jbino.com
J.Bio.Innov2(5),pp:240-259,2013 www.jbino.com
J.Bio.Innov2(5),pp:240-259,2013 www.jbino.com
J.Bio.Innov2(5),pp:240-259,2013 www.jbino.com
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