The document summarizes topics related to transgenic animals and gene therapy. It discusses transgenic cows, sheep, poultry, and fish. For each animal, it describes the process used to create transgenic versions, including pronuclear microinjection and somatic cell nuclear transfer. Benefits include producing human therapeutic proteins and altering milk composition. Challenges include high costs and low success rates. Gene therapy techniques like viral vectors and electroporation are explained for inserting genes into tissues to treat disease. Somatic gene therapy aims to modify individual patients while germline gene therapy alters heritable genes passed to offspring.

1. SEMINAR
TOPICS
Transgenic animals (cow, sheep, poultry, fish)
Gene therapy
Presented by
Snehasish
Kundu.

2. What is Gene transfer ?
The insertion of unrelated genetic information in the form of DNA into cells. Some of
these
methods involve the use of a vector such as a virus that has been specifically modified so
it can take the gene along with it when it enters the cell.
What is Transgenic animal ?
Transgenic animals are animals (most commonly mice) that have had a foreign gene
deliberately inserted into their genome. Such animals are most commonly created by the
microinjection of DNA into the pronuclei of a fertilized egg which is subsequently
implanted into the oviduct of a pseudo pregnant surrogate mother.

3. Definition- A transgenic animal is one whose genome has been altered by the transfer of a
gene or genes from another species or breed.
Discovery- The first successful transfer of embryos was achieved by Walter Heape in Angora
in
1891.
The first genetic modification of animals was reported in
1974 by the virologist Rudolph Jaenisch, then at the
Salk Institute,
The photo of two transgenic mice positioned either side of a plain mouse. The transgenic mice
have been genetically modified so that they carry a green fluorescent protein which glows green
under blue light.

4. Importance-
 Transgenic animals are routinely used in the laboratory as models in biomedical research.
They are important tools for researching human disease, being used to understand gene
function in the context of disease susceptibility, progression and to determine responses to a
therapeutic intervention.
 Transgenic farm animals are also being explored as a means to produce large quantities of
complex human proteins for the treatment of human disease. Such therapeutic proteins are
currently produced in mammalian cell-based reactors.
 A cheaper option would be to develop a means to produce recombinant proteins in the milk,
blood or eggs of transgenic animals.
 The first is human antithrombin III, a therapeutic protein produced in the milk of transgenic
goats, which is used to prevent clots in patients with hereditary antithrombin deficiency
receiving surgery or undergoing childbirth.

5.  A recombinant human C12 esterase inhibitor produced in the milk of transgenic rabbits. This is
used to treat hereditary angioedema, a rare genetic disorder which causes blood vessels in the
blood to expand and cause skin swellings.
Disadvantage- a) Transgenic animal project is extremely expensive.
b) Generation of transgenic animals are also expensive, because of long gestation
period, litter size and higher maintenance cost of the recipient animals.
c) Large number of recipients is required for embryo transfer because of low
transgenesis rate.
d) Transgenic foods have been produced and offer better productivity in terms of
both yield and quantity. However, there are some apprehension about the
safety
of transgenic foods.

6. Transgenic cow
 Definition- Transgenic cows are genetically modified (GM) cows.
They have an extra gene or genes inserted into their DNA.
The extra gene may come from the same species or
from a different species.
 Techniques used to make transgenic cows- Making a transgenic cow is a multi-step process.
1. First, the gene for the desired product is identified and sequenced. Then a gene
construct containing this desired gene is created using DNA cloning, restriction
enzyme digests and ligation.
2. The gene construct is then introduced into female bovine (cow) cells by
transfection
3. Transgenic bovine cells are selected and fused with bovine oocytes that have had
all of their chromosomes removed.

7. 4. Once fused with the oocyte, the transgenic cell’s chromosomes are
reprogrammed to direct development into an embryo, which can be implanted
into a recipient cow.
5. After a 9-month gestation period, a female calf is born. She will only express
the
transgene in her milk during lactation after her first calf is born. This is
because
expression of the transgene is controlled by a promoter specific to lactating
mammary cells.
This article gives further information on the techniques used to make transgenic cows.
 Advantages- 1. Making therapeutic proteins
Transgenic cows can be used as ‘bio factories’ to produce human therapeutic
proteins (proteins that are used to treat diseases).

8. At AgResearch, they’ve generated cows that produce human myelin basic protein.
Treatment with human myelin basic protein may help reduce the symptoms of
multiple
sclerosis.
2. Making functional foods
Milk composition can be altered to make a functional food. For example, researchers
have shown that minor proteins in milk (such as lactoferrin) could protect humans
against infection and improve gut health.
3. Hypo-allergenic milk
In 2012, the AgResearch team published the news that they had bred the first cow
in
the world to produce milk with reduced amounts of beta-lactoglobulin (BLG). BLG
is a
milk protein largely responsible for infants’ allergic reactions to cow’s milk.

9. Transgenic sheep
 Definition- In sheep, pronuclear microinjection of several hundred
copies of the foreign gene into embryos is the only published
method used to regularly produce transgenic sheep.
 Procedure to making Transgenic sheep-
1. Individual donor cells are fused to an enucleated oocyte with short-duration electric pulse.
2. e.g.: two 2.5 kilovolt /cm pulses for 10microseconds.
3. Used to fuse adult cattle fibroblasts with enucleated oocytes.
4. The pulses simultaneously induce cell fusion and oocyte activation.
5. Blastocyst stage before transferred into the uterus of a pseudopregant female.
6. Confirmed transgene at the time of birth.

11. 7. Surviving animals produced by nuclear transfer are healthy.
8. There, is a substantial loss of individual before and after birth some of the cloned animals
display abnormalities.
9. Abnormalities such as increased birth weight.
10. DNA methylation and histone modification of the original donor cell is inappropriate
maintained in the cells of the recipient animals.
 Advantages- 1. Site-specific genetic changes are possible
2. All offspring are transgenic In DNA microinjection,
 Limitation- 1. transgene integration occurs at random sites
2. Unstable things are formed
3. Expression is always constrained(limit)
4. No. of generations required

12. Transgenic poultry
Definition- In addition to retroviral vectors, microinjection of
DNA has been demonstrated to produce transgenic
poultry. Injection of DNA into the pronucleus of the
newly fertilized egg is a common procedure for the
production of transgenic

13. Procedure-

14. Transgenic fish
 Definition- A transgenic fish is one that contains genes from another species.
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.
 Method to production of Transgenic fish-
1. Microinjection method:
Microinjection method has been used successfully in the production of transgenic fish and is
a
commonly used technique due to its simplicity and reliability. Microinjection that allows
delivery of the transgene directly into the nucleus, Transgene is directly microinjected into
the
male pronuclei of fertilized eggs.

15. 2. Electroporation method:
Electroporation has been shown to be the most effective means of gene transfer in fish since a
large number of fertilized eggs can be treated in a short time by this method.
Electroporation utilizes a series of short electric pulses to permeate the cell membrane that
make possible the formation of temporary pores on the surface of the target cells through
which the transgene is introduced into the cytoplasm where it is then delivered to the
nucleus
by the cellular machinery.
3. Sperm mediated gene transfer method:
Spermatozoa are capable of binding DNA and carrying it into an egg. Fish spermatozoa can
be
stored in seminal plasma with little loss of viability for long periods. Therefore, this
technique

16. 4. Retroviral infection method:
The successful use of Retroviruses for gene delivery in fish has been reported.
 Advantage of Transgenic fish-
1. Transgenic fish have been developed for applications such as the production of human
therapeutics, experimental models for biological research, environmental monitoring, ornamental
fish and aqua cultural production.
2. Growth enhanced transgenic fish have improved feed-conversion efficiency, resulting in
economic and potential environmental benefits such as reduced feed waste and effluent from fish
farms.
3. Transgenic strains provide useful model systems for studying the consequences of growth
enhancement from genetic, physiological and ecological standpoints.
4. Commercial production of transgenic fish able to transmit desirable characteristics, such as
enhanced growth or disease resistance to their progeny.

17. Gene Therapy
 Definition- Gene therapy is the insertion of genes into an individual's cells and tissues to
treat a disease, and hereditary diseases in which a defective mutant allele is replaced
with a functional one.
 Classification- Gene therapy may be classified into two types:
1. Somatic:
In somatic cell gene therapy (SCGT), the therapeutic genes are transferred into any cell
other than a gamete, germ cell, gametocyte, or undifferentiated stem cell. Any such
modifications affect the individual patient only, and are not inherited by offspring.
Somatic gene therapy represents mainstream basic and clinical research, in
which therapeutic DNA (either integrated in the genome or as an external episome or
plasmid) is used to treat disease.

18. 2. Germline:
In germline gene therapy (GGT), germ cells (sperm or egg cells) are modified by the introduction of
functional genes into their genomes.
Modifying a germ cell causes all the organism's cells to contain the modified gene. The
change is therefore heritable and passed on to later generations.
 Vectors- Vectors in gene therapy
The delivery of DNA into cells can be accomplished by multiple methods. The two major classes are
recombinant viruses (sometimes called biological nanoparticles or viral vectors) and naked DNA or
DNA complexes (non-viral methods).
1. Viral vectors: In order to replicate, viruses introduce their genetic material into the host cell,
tricking the host's cellular machinery into using it as blueprints for viral
proteins.
Retroviruses go a stage further by having their genetic material copied into the
genome of the host cell.

19. A number of viruses have been used for human gene therapy, including retroviruses,
adenoviruses, herpes simplex, vaccinia, and adeno-associated virus.
2. Non-viral vectors: Non-viral methods present certain advantages over viral methods, such
a large scale production and low host immunogenicity.
Methods for non-viral gene therapy include the injection of naked DNA, electroporation, the gene
gun, sonoporation, magnetofection, the use of oligonucleotides, lipoplexes, dendrimers, and
inorganic nanoparticles immunogenicity.
• Injection of naked DNA- It is the process to inject the naked DNA in to the skeletal muscle and
produce a lot of excitement and lead to the possibility or using the gene as vaccines.
• Electrophoration- The last few years has seen a marked increase in the number of studies
employing intramuscular or intradermal injection of naked DNA followed by electroporation.

20.  Gene gun- In genetic engineering, a gene gun or a biolistic particle delivery system, originally
designed for plant transformation, is a device for delivering exogenous DNA (transgenes) to
cells. The payload is an elemental particle of a heavy metal coated with DNA (typically plasmid
DNA).
 Lipoplex- Plasmid DNA can be covered with lipids in an organized structure like a micelle or a
liposome. When the organized structure is complexed with DNA it is called a lipoplex. There
are three types of lipids, anionic (negatively charged), neutral, or cationic (positively charged).
Initially, anionic and neutral lipids were used for the construction of lipoplexes for synthetic
vectors.
 Sonoporation- Sonoporation, or cellular sonication, is the use of sound (ultrasonic frequencies)
for modifying the permeability of the cell plasma membrane. This technique is usually used in
molecular biology and non-viral gene therapy in order to allow uptake of large molecules such
as DNA into the cell, in a cell disruption process called transfection or transformation.
Sonoporation is under active study for the introduction of foreign genes
in
tissue culture cells, especially mammalian cells.

21. THANK YOU