It's include all the details about the transgenic technology.all the techniques like micro injection,SCNT,pro nuclear injection method.It include all the Transgenic mice bird and fish.

1. T.KASTHURI KEERTHANA
II M.SC MICROBIOLOGY
20201232516108
PG AND RESEARCH DEPARTMENT OF
MICROBIOLOGY
DR.g.ramanathan
Assistant proffessor
PG AND RESEARCH DEPARTMENT OF MICROBIOLOGY
SRI PARAMAKALYANI COLLEGE
ALWARKURUCHI

2. SEMINAR ON BIOTECHNOLOGY
SUBMITTED TO
G.RAMANATHAN
ASSISTANT PROFFESSOR

3. T.KASTHURI KEERTHANA
II M.Sc MICROBIOLOGY

6.  Trangenic animals are the animals with the modified genome. A foreign gene is
inserted into the genome of the animal to alter its DNA. This method is done to
improve the genetic traits of the target animal.
 Initially, the improvement of genetic traits was done by selective breeding methods. In
this, the animals with desired genetic characteristics were mated to produce an
individual with improved genetic characteristics. Since this technique was time-
consuming and expensive, it was later replaced by recombinant DNA technology.
 Transgenesis is the phenomenon in which a foreign gene with desired characteristics
is introduced into the genome of the target animal. The foreign gene that is introduced
is known as the transgene, and the animal whose genome is altered is known as
transgenic. These genes are passed on to the successive generations.
 The transgenic animals are genetically engineered and are also known as genetically
modified organisms. The first genetically modified organism was engineered in the
year 1980.

7. Physical Transfection
 In this method, the gene of interest is directly injected into the pronucleus of a fertilized ovum. It is the very
first method that proved to be effective in mammals. This method was applicable to a wide variety of species.
Other methods of physical transfection include particle bombardment, ultrasound and electroporation.
Chemical Transfection
 One of the chemical methods of gene transfection includes transformation. In this method, the target DNA is
taken up in the presence of calcium phosphate. The DNA and calcium phosphate co-precipitates, which
facilitates DNA uptake. The mammalian cells possess the ability to take up foreign DNA from the culture
medium.
Retrovirus-Mediated Gene Transfer
 To increase the chances of expression, the gene is transferred by means of a vector. Since retroviruses have the
ability to infect the host cell, they are used as vectors to transfect the gene of interest into the target genome.
Viral Vectors
 Viruses are used to transfect rDNA into the animal cell. The viruses possess the ability to infect the host cell,
express well and replicate efficiently.
Bactofection
 It is the process by which the gene of interest is transferred into the target gene with the help of bacteria.

8. Dolly Sheep
 Dolly the sheep was the first mammal to be cloned from an adult cell. In this, the udder cells
from a 6-year-old Finn Dorset white sheep were injected into an unfertilized egg from a
Scottish Blackface ewe, which had its nucleus removed. The cell was made to fuse by electrical
pulses. After the fusion of the nucleus of the cell with the egg, the resultant embryo was
cultured for six to seven days. It was then implanted into another Scottish Blackface ewe
which gave birth to the transgenic sheep, Dolly.
Transgenic Mice
 Transgenic mice are developed by injecting DNA into the oocytes or 1-2 celled embryos taken
from female mice. After injecting the DNA, the embryo is implanted into the uterus of
receptive females.

9.  Although the human is the mammal we are generally most interested in learning
more about, it is also the one animal we cannot use for genetic experiments for
obvious ethical reasons
 Mice naturally develop conditions that mimic human disease, such as
cardiovascular disease, cancer and diabetes
 Mouse are a favorite model for human disease because it has a relatively low cost
of maintenance and a generation time that measures only nine weeks
 Developments in molecular biology and stem cell biology have allowed
researchers to create custom-made mice through gene targeting in mouse
embryonic stem (ES) cells
 Certain diseases that afflict only humans, such as cystic fibrosis and Alzheimer's
can also be induced by manipulating the mouse genome and environment

10.  Embryonic stem (ES) cells are pluripotent cell lines
with the capacity of self-renewal and a broad
differentiation plasticity
 They are derived from pre-implantation embryos
and can be propagated as a homogeneous,
uncommitted cell population for an almost unlimited
period of time
 Even after extensive genetic manipulation, mouse
ES cells are able to reintegrate fully into viable
embryos when injected into a host blastocyst
 After these pre-implantation embryos are implanted
into a surrogate mother, they develop into mosaic
offspring known as chimeras. The tissues of chimeric
mice are comprised of a mixture of cells that
originated from both the host embryo and the ES
cells.

11.  A knockout mouse is a genetically engineered mouse in which one or more genes
have been turned off through a gene knockout
 Important animal models for studying the role of genes which have been
sequenced, but have unknown functions
 By causing a specific gene to be inactive in the mouse, and observing any
differences from normal behaviour or condition, researchers can infer its probable
function.
 Gene knockout is a technique for selectively inactivating a gene by replacing it
with a mutant allele in an otherwise normal organism (mice)
 Knockout mice are a useful model system for studying certain human genetic
diseases.

14.  Mutant alleles are introduced by
homologous recombination into Embryonic
Stem cells
 ES cells containing the knockout mutation
are introduced into early mouse embryos.
The resultant mice will be chimeras
containing tissues derived from both the
transplanted ES cells and host cells. These
cells can contribute to both germ cell and
somatic cell population
 Chimeric mice are mated to assess
whether the mutation is incorporated into
the germline
 Chimeric mice each heterozygous for the
knockout mutation are mated to produce
homozygous knockout mice

19.  Fish are of particular interest to researchers since many fish produce large
quantities of eggs; those eggs, being external to the animal (as opposed to
mammals that produce fewer eggs internally), make it relatively simple to insert
novel DNA
 Research on transgenic fish is currently under development for at least 35 species
of fish worldwide, as well as for a variety of mollusks, crustaceans, plants, and
marine microorganisms, for various purposes.
 Transgenic technology has been successfully used to develop fast-growing super-
fish stocks for human consumption,
 to produce pharmaceuticals,
 to test water contamination in both developed and developing countries.
 Several laboratories now have GM fish with increased growth performance caused
by extra copies of GH genes

20.  For the aquacultural importance, Indian major carps, Common carp, Channel
catfish, Chinese carps, Salmon, Trout and Tilapia are the best species for the
transgenic project.
 Improvement of growth rate, imparting disease and environmental stress
resistance are some important traits for transgenesis.
 Reporter gene is defined as a gene whose products detects or marks the cells,
tissues, organisms that express the gene from those that do not.
 Reporter genes isolated from prokaryotes, E. coli, are used in fishes- lac Z gene,
Cat (Chloramphenicol Acetyl Transferase gene).
 Recently luciferase and green fluorescent protein are used.

21.  Growth is a complex biological process involving genetic, hormonal, nutritional
and environmental factors.
 ‘Growth hormone’ (GH) is produced by the anterior lobe of the pituitary plays the
key role.
 It increases growth by stimulating appetite and improving the food conversion
efficiency.
 GH is a protein hormone having a molecular weight of about 22 kilodaltons.
 Transgenic fish carrying GH gene will produce growth hormone endogenously by
passing the necessity of exogenous hormone treatment.
 GH gene has been cloned in some fishes either from the genomic library or from
the cDNA library.

22.  Production of cold resistant fish variety is useful for establishing aquaculture
industry in the temperate region, where water gets frozen during winter.
 The gene responsible for imparting cold resistance was cloned from winter
flounder (Pseudopleuronectes americanus), which lives in the polar sea.
 This species avoids freezing of its blood even at –7C temperatures by producing a
set of anti-freeze proteins (AFP).
 AFP are produced in the liver and exported to the blood stream.

23.  Fish has poorly developed antibody dependent immunity.
 Efforts to produce disease resistance in fish stocks by transgenesis have begun
recently.
 The potential of Rainbow trout lysozyme gene as a bacterial inhibitor was
assessed in Atlantic salmon.

25. Glofish
 The GloFish is a patented brand of genetically modified (GM) fluorescent
zebrafish with bright red, green, and orange fluorescent color.
 The transgenic zebrafish (Danio rerio) expressing a red fluorescent protein from a
sea anemone under the transcriptional control of the promoter from the myosin
light peptide 2 gene of zebrafish.

26.  GM food safety depends on the nature of the gene, the transgene product it encodes and the
resulting phenotype.
 Ethics and animal protection concerns allows the development of healthy and safe fish only.
 Transgenic fish have received extra copies of GH genes, resulting in only moderately raised
levels of circulating GH.
 GH is a protein hormone which is degraded along with all other food protein.
 Meat from fish modified with GH is regarded as completely safe for human consumption
Concerns have been voiced of the possible risks of consumption of transgenes,
 their resulting protein,
 potential production of toxins by aquatic transgenic organisms,
 changes in the nutritional composition of foods,
 activation of viral sequences and
 allergenicity of transgenic products

27.  The possible impacts from the escape of GM organisms from aquaculture facilities are of great
concern to some scientists and environmental groups.
 Critics and scientists predict that GM fish could breed with wild populations of the same species
and potentially spread undesirable genes
 Escaped transgenic fish could harm wild fish through increased competition or predation.
 The consequences of such competition would depend on many factors, including the size of the
wild population, the number and specific genetic strain of the escaped fish, and local
environmental conditions.
 FDA could require that only sterile GM fish be approved for culture in ocean pens.
 Fertilized fish eggs that are subjected to a heat or pressure shock retain an extra set of
chromosomes.
 The resulting triploid fish do not produce normal eggs or sperm, and females do not exhibit
maturation of the ovary or reproductive behaviors.
 Thus, all-female lines of triploid fish are the best current method to ensure non-breeding
populations of GM fish.

28.  Biotechnology proponents maintain that genetic modification has many advantages over
traditional breeding methods, including faster and more specific improvement of beneficial traits.
 Genetic modification allows scientists to precisely select traits for alteration, enabling them to
create an organism that, for example, grows larger or faster or has a different nutritional content.
 Increased freeze resistance in fish could lead to the ability to grow freeze-resistant species in
previously inhospitable environments, allowing aquaculture to expand into previously unsuitable
areas.
 Biotechnology proponents claim these advantages could translate into a number of potential
benefits, such as reduced costs to producers, lower prices for consumers for edible fish and
pharmaceuticals, and environmental benefits, such as reduced water pollution from wastes.
 M.ajority of consumers in the United States appear to have accepted GM food and feed crops, it is
uncertain whether consumers will be as accepting of GM fish

29.  Avian ova are normally fertilized approximately 30 minutes after ovulation. Cell division occurs in the oviduct
for approximately 20 hours before ovi position. At this time, the embryo is comprised of approximately 60,000
pluripotent cells, which are collectively called the blastoderm.
 The presence of a large yolk and multiple pronuclei makes direct microinjection of DNA impractical.
 Therefore, DNA microinjection into fertilized bird eggs to produce transgenic strains is not possible;
 During fertilization in birds several sperms can penetrate the ovum, instead of only one as in case of mammals.
 Therefore, it is not possible to identify the male pronucleus that will fuse with the female pronucleus.
 Microinjection of DNA into cytoplasm is not enough for the process to proceed as the DNA will not integrate
into the genome of the fertilized egg.
 The technique also would be difficult as the avian ovum after fertilization become enveloped in tough
membrane and surrounded by large quantities of albumin and enclosed in inner and out shell membranes.

30. WHAT CAN WE USE
TRANSGENIC CHICKEN FOR?
 To improve the genetic makeup of the existing strains with respect
to
 resistance to avian viral and coccidial diseases,
 better feed efficiency,
 lower fat and cholesterol in eggs and
 better meat quality.
 The egg with its high protein content could be used as a source of
pharmaceutical proteins

32.  https://www.springer.com/journal/11248
 https://link.springer.com/journal/11248/volumes-and-issues
 https://pubmed.ncbi.nlm.nih.gov/22833168/
 https://scholar.google.co.in/scholar?q=recent+research+on+transgenic+methods&h
l=en&as_sdt=0&as_vis=1&oi=scholart

33.  https://www.youtube.com/watch?v=ujZHrR1mro8
 https://youtu.be/TJu8sWZDhU4
 https://www.youtube.com/watch?v=EFNtTfhhQ70
 https://youtu.be/KG1mk6PIE4s