The document discusses transgenic mice and fish. It defines transgenic animals as those permanently engineered through gene insertion. Methods to create transgenic mice include retroviral vectors, DNA microinjection into pronuclei, and using engineered embryonic stem cells. Transgenic mice have aided research in many areas. Transgenic fish are also discussed, including important salmon and tilapia examples. Gene flow and food safety are concerns for transgenic animals.

1. TRANSGENIC ANIMALS:
MICE AND FISH
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By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. CONTENTS
• Introduction.
• Definition.
• Importance of transgenic animals.
 Transgenic mice
• Methods for introducing a foreign gene:
– The retroviral vector method
– The DNA microinjection method/ pronuclear microinjection
– Genetically engineered embryonic stem cells
 Transgenic fish
 What is transgenic fish?
 A few facts to know to know about transgenic fish.
 Important points needed for genetic engineering (gene transfer) to produce
transgenic fish.
 Development of transgenic fishes.
 A few examples
 Auto-transgenesis.
 Controlled culture of transgenic fish and feed.
 Gene transfer technology for development of transgenic fishes.
• Gene flow.
• Food safety issues.
• Conclusion.
• Bibliography.
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3. DEFINITION
 “Transgenesis may be defined as the introduction of exogenous (foreign) DNA into
the genome, such that it is stably maintained in a heritable manner.”
 Animals that have been permanently engineered by gene insertion are called
transgenic animals.
 Any foreign gene that are added are called transgenes.
 A genetically modified organism is one that had its genetic material altered through
any method, including conventional breeding. A genetically engineered organism is
one that has been genetically modified using recombinant DNA technology.
 A transgenic organism has been genetically engineered using a foreign gene, usually
belonging to a different species.
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4. FIRST TRANSGENIC ANIMAL
 The first transgenic animal was produced in 1981 by Ralph
Brinster and Richard Palmiter.
 They succeeded in introducing a gene for rat growth
hormone into the fertilized egg of mice. The injected DNA
was constructed so as to contain the protein of rat growth
hormone gene in a position just downstream from the
promoter region of the mouse metallothionein gene.
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6. IMPORTANCE OF
TRANSGENIC ANIMALS
• To study normal physiology and development: gene expression and
developmental processes
• To study disease.
• To obtain biological
– Eg: Transgenic sheep (milk): in sheep, scientists have introduced alpha-1-
antitrypsin whixh is used to treat emphysema.
– Transgenic cow “rosie”: alpha lactalbumin human protein to substitute of
mother’s milk to infants.
• Vaccine safety tests.
• To check toxicity of chemicals.
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7. Transgenic Mice
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8. • Mouse is the animal of choice for transgenic experiments and the reasons
behind this choice are:
– a small animal,
– it can be easily handled,
– and mouse is regarded as researcher-friendly by biotechnologists.
– it produces more eggs (normal mouse5 -10 eggs, super ovulated
mouse can yield up to 40 eggs) unlike the large domestic animals.
• In the last two decades, hundreds of different genes have been introduced
into the various mouse strains.
• Transgenic mice have significantly contributed to the understanding of
molecular biology, genetics,immunology and cancer, besides creating
animal models for several human genetic diseases.
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10. METHODS FOR INTRODUCING
A FOREIGN GENE
1. Introduction of retroviral vectors in the cells of
an early stage embryo.
2. Pronuclear microinjection.
3. Introduction of genetically engineered
embryonic stem cells into an early stage
developing embryo.
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11. THE RETROVIRAL VECTOR METHOD
The retroviral vector method is an effective
means of integrating the transgene into the
genome of a recipient cell.
Retroviruses have RNA genomes that areused as
templates for reverse transcriptase to synthesize
a DNA copy that can be inserted into the host cell
genome.
However, vectors derived from these viruses can
transfer only small pieces (~8 kilobases [kb]) of
DNA.
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12. MAJOR DRAWBACK OF THE
RETROVIRAL VECTOR
The genome of the retroviral strain (helper
virus) that is needed to create large quantities
of the vector DNA can be integrated into the
same nucleus as the transgene.
Transgenes introduced on some retroviral
vectors are silenced in mouse embryos.
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13. THE DNA MICROINJECTION METHOD/
PRONUCLEAR MICROINJECTION
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14. LIMITATIONS OF MICROINJECTION
METHOD
• Low efficiency.
• The foreign DNA randomly integrates into the host genome.
• Sometimes, even many pieces of DNA get incorporated at a
single site. Further, transgenes may not be expressed at all
or sometimes under expressed or, even overexpressed.
• Microinjection procedures are time consuming, costly and
labor intensive.
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15. Advantages of Microinjection Technique
Disadvantages of Microinjection
Technique
(1) Optimum quantity of DNA can be
delivered per cell, increasing chances for
integrative transformation.
(1) A single cell can be injected at a time,
hence it is time consuming process.
(2) The delivery of DNA is precise, even
into nuclei of target cell again improving
chances for integrative transformation.
(2) It requires sophisticated instruments
and specialized skills.
(3) The small structure can be injected.
(3) Limited embryonic time restricts
injection to more eggs and a low
transformation rate.
(4) It is a direct physical approach; hence
it is a host range independent.5/15/2020 15

16. THE ENGINEERED EMBRYONIC STEM
CELL METHOD
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17. Selection of transgenic cells
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18. TRANSGENIC MICE: APPLICATIONS
Transgenic Disease
Models: Alzheimer
Disease
Using Transgenic Mice as
Test Systems
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19. Transgenic
Fish
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20. WHAT IS A TRANSGENIC FISH?
 A transgenic fish is an improved variety of fish provided with one or more
desirable foreign gene for the purpose of enhancing fish quality, growth, resistance
and productivity.
 A FEW FACTS TO KNOW ABOUT TRANSGENIC FISH:
 Typically, genes of one or more donor-species are isolated, and spliced into
artificially constructed infectious agents, which act as vectors to carry the genes
into the cells of recipient species. Once inside a cell, the vector carrying the genes
will insert into the cell’s genome.
 To date, transgenes have been introduced by microinjection or electroporationof
DNA into the fertilized eggs of a number of fish species, including carp,catfish,
trout, salmon, arctic char, and tilapia.
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21. • The pronuclei of fish are notreadily seen under a microscope after fertilization; therefore,
linearized transgene DNA is microinjected into the cytoplasm of either fertilized eggs or
embryos that have reached the four-cell stage of development.
• Unlike mammalian embryogenesis, fish egg development is external; hence, there is no need
for an implantation procedure.
• Development of transgenic fish occurs in temperature-regulated holding tanks.
• The survival of fish embryos after DNA microinjection is high (35 to 80%), and the production
of transgenic fish ranges from 10 to 70%.
• The presence of a transgene is scored by PCR analysis of either nucleated erythrocytes or
scale DNA.
• Founder fish are mated to establish true-breeding transgenic lines.
• One of the most important aspects between fish and other terrestrial animals for cultivation
and genetic improvement is that, usually, fishes have higher levels of genetic variation and
hence more scopes for selection than most mammals or birds.
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22. IMPORTANT POINTS NEEDED FOR GENETIC
ENGINEERING (GENE TRANSFER) TO PRODUCE
TRANSGENIC FISH
(1) A gene sequence is to isolate for the particular characteristics; for example, growth
hormone gene.
(2) These genes (gene sequence) are then inserted into a circular DNA known as plasmid
vector (enzymes endonucleases and ligases are used).
(3) Plasmids are harvested in the bacteria to produced billions of copies.
(4) Plasmids are introduced into linear DNA. The linear DNA is sometimes called a gene
cassette because it contains several sets of genetic material in addition to new inserted
gene; for example, growth hormone gene. The technology is available to integrate genes
in germ line of developing individual (fish) and finally transmitted into further
generations.
(5) Making the cassette a permanent part of fish’s genetic makeup.
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23. TRANSGENIC SALMON
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24. TRANSGENIC
MEDAKA
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25. TRANSGENIC TILAPIA
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26. AUTO-TRANSGENESIS
CONTROLLED CULTURE
OF TRANSGENIC FISH
AND FEED
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27. APPLICATIONS OF TRANSGENIC FISH
(1) For increasing fish production to meet the growing due to demand of food due
to increase in world population.
(2) For production of pharmaceutical and other industrial products from piscine
origin.
(3) For development of transgenic native glow fish varieties for aquarium.
(4) As fish biosensors for monitoring aquatic pollution.
(5) For isolation of genes, promoters and synthesis of effective gene constructs.
(6) For researches in embryonic stem cells and in-vitro embryo production.
(7) For production of anti-freeze protein.
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28. GENE FLOW
• One of the larger environmental concerns raised by transgenic animals is the possibility that a transgenic
species raised in open will escape and spread novel traits into the ecosystem by breeding with wild
relatives, a biological process known as “gene flow.”
• Gene flow between transgenic or conventionally animals and wild populations is an environmental
concern, because it may present a threat to natural biodiversity.
• Some researchers believe that the genetic differences introduced to a transgenic animalmay impact its net
fitness, a scientific term meaning an organism’s ability to survive and pass its genes to future generations.
• Essentially mating success would ensure the spread of the novel gene throughout the population, but the
inability to survive would reduce the population size of subsequent generations and potentially lead to
extinction.
• A declining population would also have secondary impacts on other species that feed on, or otherwise
depend on it. Populations unable to’ successfully switch over to another food source, or those whose
survival or reproduction depends directly on the declining population, would also suffer.
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29. FOOD SAFETY ISSUES
• One important food safety issue involves the extent to which fish absorbs and
stores environmental toxins, such as mercury, high levels of which could pose a
danger to humans who eat the contaminated fish.
• Some scientists’ worry that discrete biological changes induced by the genetic
engineering process might enable transgenic fish to absorb a toxin that
conventional fish cannot absorb or to better tolerate higher levels of a toxin
already known to cause concern.
• Some scientists have expressed concern that the genetic engineering process
could increase the allergic potential of fish, particularly through the introduction of
novel proteins that never before existed in the food chain.
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30. CONCLUSION
• Transgenic animals have contributed a lot to human welfare which can be grouped into areas
as:
– Agriculture- i.e . for breeding, quality enhancement , and disease resistance.
– Medicine- for xenotransplantation, for providing nutritional supplements and
pharmaceuticals and for human gene therapy.
– In the field of industries.
• Interestingly, the creation of transgenic animals has resulted in a shift in the use of laboratory
animals — from the use of higher-order species such as dogs to lower-order species such as
mice — and has decreased the number of animals used in such experimentation, especially in
the development of disease models. This is certainly a good turn of events since transgenic
technology holds great potential in many fields.
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31. BIBLIOGRAPHY
BOOK
• MOLECULAR BIOTECHNOLOGY- PRINCIPLES AND APPLICATIONS OF RECOMBINANAT,
4th Edition by Bernard Glick, Jack J. Pasternack and Cheryl L. Patten
INTERNET SOURCES
• http://www.yourarticlelibrary.com/fish/genetics-fish/transgenic-fishes-meaning-
development-and-application/88723
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4095860/
• http://www.actionbioscience.org/biotechnology/margawati.html
RESEARCH ARTICLES
• TANSGENIC ANIMALS: PRODUCTION AND APPLICATION, by Manmohan Singhal and
Niraj Kansara, International Journal o Pharmaceutical science and research.
• METHODS TO GENERATE TRANSGENIC ANIMALS By Louis- Marie Houdebine.
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