WHAT IS AN EMBRYO TRANSFER TECHNOLOGY

Embryo transfer is a bio-technique where embryos are collected from the donor females and
transferred in to the uterus of recipients which serves as a foster mother for its development
throughout the remainder period of pregnancy".
HISTORY OF EMBRYO TRANSFER
 1890: The first successful embryo transfer was carried out in rabbit by Heap.
 1949: First lamb was born by embryo transfer technology performed by Berry.
 1951: First calf was born by embryo transfer technology performed by Willet et. al.
 1983: Embryo transfer technology was performed in Asian buffalo by Drost et. al.

STEPS OF EMBRYO TRANSFER

i. Selection of donor animal
ii. Selection of recipient animal
iii. Superovulation (i.e. release of multiple eggs) of donor animal.
iv. Artificial insemination of donor animal
v. Embryo collection
vi. Evaluation of embryo
vii. Transfer of embryo into recipient animal
I. Selection of donor animal
 Donor should have following characteristics:
1. Superior individual performance.
2. Regular Estrus cycle.
3. Ovaries must be free (no adhesions).
4. Younger (4-8 yrs. of age).
5. Healthy and have good body weight
Il. Selection of recipient animal
 Recipient should have following characteristics:
1. Healthy, free from infection and have good body weight.
2. Regular Estrus cycle.
3. Must have good cyclic of desired stage at the time of embryo transfer.
4. Should have good milking and good mothering ability.
Ill. Superovulation of Donor animal
  Superovulation is the procedure for increasing ovulatory response by administration of
hormones (gonadotropins) to produce several ova instead of one which is normally produced
at each estrus.
 These large number of ova are later on fertilized and embryo produced can be transferred to
the other females.
 The basic principle of superovulation is to stimulate extensive follicular development through
the use of hormone preparation, which is given intramuscularly or subcutaneously, with
follicle stimulating hormone (FSH) activity.
IV. Artificial insemination of donor animal
 Artificial deposition of semen into female genital tract is known as artificial insemmation.
 Donor should be inseminated artificially by the semen of superior quality bull 2-3 times at 12
hours, 24 hours and 36 hours interval, beginning at 8-10 after the onset of estrus.
 This is required because ovulation can occur over an extended time period.
 Fresh semen is preferred.
V. Embryo Collection
• Embryo can be collected by following methods:
1. Surgical method:
 Surgical method is most often used in sheep, goat and swine through mid-ventral incision
under general anaesthesia.
 The method can be performed on day 3-4 after estrus in sheep and goat (8-cell embryo or
less).
2. Non-surgical or Trans cervical method:
 It involves two ways or three ways Foley's catheter which allows flushing fluids to pass into
the uterus and at the same time allows fluids to be returned from the uterus to a collecting
receptacle.
 A small balloon near the end of catheter can be inflated just inside the uterine horn to prevent
the flushing fluid from escaping through the cervix.
 Collection of bovine embryo should be made at 6-8 days post-breeding at compact morula or
blastocyst stage.
VI. Evaluation of embryo
 After collection and before transfer to the recipients, the embryos are evaluated under
stereomicroscope at 50 -100 X magnification. Day seven bovine embryos (compact morula or
blastocyst stage) are about 150-190 pm in diameter and are still within the zona pellucida.
 Embryos are graded based on following Characteristics:
 Compactness of the cells
 Regularity of shape
 Variation in cell size
 Color and texture of cytoplasm
 Presence of vesicles, extruded cells, cellular debris.
VIl. Transfer of embryo into recipient animal
• Recipient should be in estrus within 12 hours of the donor so that it should posses good Corpus
Luteum at the time of transfer. To maximize success rate of the transfer, the recipient's estrus should

be in sync with that of the donor.

A. Surgical method of Embryo transfer
 This method involves incision and is preferred in sheep, goat and pig. A small syringe fitted
with 21 gauge needle is used to make the transfer.
 When the embryo is placed in the uterus, the needle is carefully inserted through the wall of
uterine horn.
 When embryo is placed in oviduct then the needle is inserted through the infundibulum into
the ampulla where the embryo is deposited.
B. Non Surgical method of Embryo transfer
 In this method, flushed embryos that pass inspection are loaded into an artificial inseminated
straw.
 If the embryo is frozen it is thawed in a warm water bath (920F) for less than 30 seconds and
placed in a specially designed transfer gun and covered with a sterile sheath.
 The transfer gun inserted into the uterine horn on the side as the corpus luteum. The embryo
is deposited one third the way up the uterine horn. This method is mostly used for cattle and
mare.
ADVANTAGES OF EMBRYO TRANSFER
I. It results of increase in the number of offspring from superior females.
Il. By this technique one can obtain offspring from old or injured animals which are incapable of

breeding naturally.
Ill. There can be increase in farm income through embryo sale business.
IV. Exportation or importation of embryos is easier than with live animals.
DISADVANTAGES OF EMBRYO TRANSFER
1. It is a technique is costly technique.
Il. Success rates are less than artificial insemination.
Ill. Embryo transfer technique requires a technician with the skills to flush embryos from the
reproductive tract.
IV. There is possible risk of spreading of disease through recipients.
APPLICATIONS OF EMBRYO TRANSFER
i. Faster genetic improvement of animals.
ii. Disease control.
iii. Import and Export of embryos.
iv. Cure of infertility.
v. Twins production in cattle.
vi. Conservation of endangered species.
 What are Genes?
 Genes (which are carried on chromosomes) are the basic physical and functional units of
heredity.
 Genes are specific sequences of nucleotides bases that encode instructions on how to make
proteins.
 When genes are altered or defective then the proteins encoded by them also get defected
and are unable to carry out their normal functions which leads to genetic disorders.
 WHAT IS GENE THERAPY ?
 Gene therapy is a technique in which a "defective gene is replaced by the functional gene"
so that the body can make the functional protein and therefore eliminate the root cause of
the disease.
 A carrier molecule called a "vector" must be used to deliver the therapeutic gene to the
patient's target cells.
TYPES OF GENE THERAPY
 On the basis of target cells:
1. Somatic Gene therapy:
 When functional genes are transfer into body cells (i.e. somatic cells) other than germ cells,
is known as somatic gene therapy. Genes transferred to somatic cells are not passed to
offspring.
2. Germline Gene therapy:
 When functional genes are transfer into germ cells (i.e. sperm or egg), is known as germline
gene therapy. Genes transferred to germ cells are passed on to future generations. Generally
changes are permanent in case of germline gene therapy.
 On the basis of delivery approaches:
i. Ex vivo technique:
 Isolate cells with genetic defect from patient.
 Grow the cells in culture.
 Introduce the therapeutic gene to correct gene defect.
 Select the genetically corrected cells and grow.
 Transplant the modified cells to the patient.
ii. In vivo technique:
 The direct delivery of the therapeutic gene into the target cells of a particular tissue.
 Many tissues are the potential candidates for this approach.
 E.g. liver, muscle, skin, spleen, lung, brain and blood cells etc.

METHODS OF GENE DELIVERY

l. Virus mediated Transfer
2. Direct Transfer or Non-viral Transfer
l. Gene transfer using Viral vectors
 Virus bind to their hosts and introduce their genetic material into the host cell.
  A viral vector is a virus which has been modified in a laboratory environment for purpose of
introducing genetic material into a cell.
 To form a viral vector, remove the genes in the virus that cause disease.
 Then replace those genes with genes encoding the desired effect (for instance, insulin
production in the case of diabetics).

l. Simian Virus 40 (SV40)

 SV40 has icosahedral capsid and a circular double stranded DNA genome of approx. 5 kb.
 The genome has 2 transcription units early and late regions that face in opposite directions.
 The early region produces regulatory proteins, while the late region produces components
of the viral capsid.
 During the first stage of the SV40 infection cycle, the early transcript produces two proteins,
known as the large T and small t tumor antigens.
 The function of the T antigen (protein) is to binds to the viral origin of replication and is
absolutely required for genome replication.
 All SV40 vectors must have a functional T antigen otherwise they cannot replicate.
 SV40 Vectors
 Either the late or early region of SV 40 virus could be replaced with foreign DNA.
 Since both these regions are essential for the infection cycle, their functions had to be
provided in trans initially by a co-introduced helper virus.
 Major problem only 2.5 kb of foreign DNA Incorporated.
Il. Adeno Virus
 Adeno viruses are non-enveloped DNA viruses, linear genome and double stranded DNA
molecule of about 36kb.
 Genome Regions: distinguished into Early (E), and Late (L) transcription regions.
 Adenoviral vectors have a wide range of action and are able to deliver nucleic acids to both
dividing and nondividing cells
Ill. Vaccinia Virus
 The virus has a complex structure and a large double stranded linear DNA genome (—300
kb). Unusual for a DNA virus, the it replicate in the cytoplasm of the infected cell rather than
its nucleus.
 Direct ligation vectors are developed that are transfected into cells containing a helper virus
to supply replication and transcription enzymes in trans.
 Transgene expression needs to be driven by an endogenous vaccinia promoter, since
transcription relies on proteins supplied by the virus.
 Sequence TTTTTNT must be removed from all DNA sequences expressed in vaccinia vectors,
since the virus uses this motif as a transcriptional terminator.
IV. Herpes Simplex Virus
 Herpes simplex viruses (HSV) consists Structure of the
Herpesvirus virion of a relatively large linear DNA genome of
double-stranded DNA (150kb), encased within an icosahedral
protein cage called the capsid, which is wrapped in a lipid
bilayer called the envelope. The envelope is joined to the
capsid by means of a tegument. This complete particle is
known as the virion.
 RNA Viral Vectors
 l. Retrovirus
 Retroviruses are a class of enveloped viruses that contain two copies of
single strand sense (+) RNA genome that replicates via double stranded
DNA intermediate.
 Viral genome is 7—10 kb in size, containing at least three genes: gag,
POI, env.
 The gag gene encodes a viral structural protein pol encodes the reverse
transcriptase and Retrovirus integrase, and the env gene encodes viral
envelope proteins.
2. NON-VIRAL VECTOR SYSTEMS/DIRECT TRANSFER
METHODS
S. No. METHOD OF TRANSFECTION
1. Electroporation
11. Gene gun/Biolistic/Microprojectile/ bombardment
111. Microinjection
1.ELECTROPORATION
• Electroporation, is a significant increase in the permeability of the cell plasma membrane
caused by an externally applied electrical field. Hence created pores in the cell membrane.
• DNA is then move inside the cells through these pores.
• It is generally accepted that for a given pulse duration and shape, a specific transmembrane
voltage threshold exists for the manifestation of the electroporation phenomenon(from
0.5 V to 1 V).
2. GENE GUN
• This method uses accelerated micro projectiles to deliver DNA into intact tissues and cells. It
is also called as Biolistic/Microprojectile bombardment
• DNA is deposited on the surface of gold particles, which are then accelerated by pressurized
gas and expelled onto cells or a tissue. The momentum allows the gold particles to penetrate a few
millimeters deep into a tissue and release DNA into cells on the path.
3. DNA MICROINJECTION
• Direct transfer of DNA into the cell without a carrier is called DNA microinjection. This can
only be done for only few cells at a time.
• This technique is used mainly for large cells such as oocytes.
• The DNA can be directly injected into tissues, such as skin, muscle or internal organs or it
can be injected into the blood.
APPLICATIONS OF GENE THERAPY
l. GENE THERAPY IN CANCER
Strategies:
i. Gene therapy with Antisense oligonucleotide:
Antisense RNA hybridizes with mRNA and inhibit the gene expression in cancer cells.
ii. Gene therapy with tumor suppressor gene:
• Reintroduction and expression of wild-type tumor suppressor p53 gene into p53 altered tumor cells.

2. GENE THERAPY IN CARDIOVASCULAR DISEASE

Thrombosis is formation of blood clot in blood vessels. Thrombolysis is the dissolution of a blood
clot formed in blood vessels. So thrombolysis can be done by transferring tissue-type plasminogen
activator (tPA) gene.

3. GENE THERAPY IN ALZHEIMER'S DISEASE

Alzheimer's disease is a progressive disorder that causes brain cells to degenerate and die. Nerve
Growth Factor (NGF) is one of the neurotrophic peptide family responsible for maintenance of
neuronal function and growth.
Defect in the Nerve Growth Factor coding gene was found in patients with Alzheimer's disease.
Gene therapy now is the promising field for patients with Alzheimer's disease .

MAJOR ACHEIVEMENTS OF GENE

THERAPY
The First Case (September 14th, 1990)
Ashanti DeSilva was treated for Sever combined immunodeficiency (a disease in
which both B and T cells are absent, hence patient is immuno compromised)
Doctors removed her white blood cells, inserted the missing gene for Adenosine deaminase enzyme
into the WBC, and then put them back into her blood stream. This strengthened her immune system.
ADVANTAGES OF GENE THERAPY
 It is treatment of a genetic disease at the root of the problem i.e. at the DNA level.
 It has potential to treat a disease for which no treatment is currently available.
 It also has the potential for life-long treatment from a single injection.

DISADVANTAGES OF GENE THERAPY

1. Short-lived nature of gene therapy: Before gene therapy can become a permanent
cure for any condition, the therapeutic DNA introduced into target cells must remain
functional and the cells containing the therapeutic DNA must be long-lived and
stable. Problems with integrating therapeutic DNA into the genome and the rapidly
dividing nature of many cells prevent gene therapy from achieving any long-term
benefits.
2. Immune response:
 Anytime a foreign object is introduced into human tissues, the The risk of stimulating the
immune system in a way that reduces gene therapy effectiveness is always a potential risk.
 Furthermore, the immune system's enhanced response to invaders it has seen before, makes it
difficult for gene therapy to be repeated in patients.
3. Problems with viral vectors:
 Viruses, while the carrier of choice in most gene therapy studies, present a variety of
potential problems to the patient - toxicity, immune and inflammatory responses, and gene
control and targeting issues.
 In addition, there is always the fear that the viral vector, once inside the patient, may recover
its ability to cause disease.
4. Multigene disorders:
Conditions or disorders that arise from mutations in a single gene are the best candidates for gene
therapy. Unfortunately, some the most commonly occurring disorders, such as heart disease, high
 blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of
variations in many genes. Multigene or multifactorial disorders such as these would be especially
difficult to treat effectively using gene therapy.
5. Costly:
Treatment using gene therapy is a costly procedure.
6. Experimental skills:
A skilled person can performed gene therapy experiments.
FUTURE PROSPECTS OF GENE THERAPY
• In future, we can identify more efficient ways to deliver genes.
• We can also develop more target-specific vectors, (which can insert genes on the precise location).
• Ensure transplanted genes are precisely controlled by the body's normal physiologic signals