Cloning, Transgenic
and knockout animals
Cloning
The process of generating a genetically identical copy
of a biological unit (e.g. a DNA sequence, cell, or
organism) from which it was derived, especially by way
of biotechnological methods.
In 1996 the first clone of an adult mammal, a female
sheep named Dolly, was born and created using
somatic cell nuclear transfer (SCNT) by Wilmut and
Campbell.
• After Wilmut’s success , a lot of cloned animals were
created , such as a cat in 2001, • rabbits in 2002 and
• dog in 2005 • Rhesus monkey in 2007
Purpose of cloning
• For medical or diagnostic purposes
• To produce animal models of disease
• Produce stem cells
• To study the DNA sequence, gene expression.
• Cloned genetically engineered animals for drug production
• Reviving endangered or extinct species
• To generate groups of genetically identical animals for research purposes.
• To rapidly propagate (Produce) desirable animal stocks.
• To improve the efficiency of generating and propagating transgenic
livestock.
• To produce targeted genetic alterations in domestic animals.
• To pursue basic knowledge about cell differentiation.
Types of cloning
• DNA or Gene Cloning : making copies
of DNA/genes - Useful for research,
making pharmaceutical and industrial
proteins, gene therapy, genetic
engineering.
• Reproductive Cloning: making copies
of a whole animal/human – useful to
propagate transgenic /genetically
modified organisms
• Therapeutic Cloning: making copies
of an embryo in order to harvest its
stem cells – useful for research or
stem cell transplants (rebuild organs)
to cure disease
Application of cloning in pharmaceutical
and medical sector
• Cloning use to produce animal models of disease to learn about human
diseases, therapeutic action of drugs.
• Breeding androgenic body tissue: Cloning use to make stem cells that
are genetically identical to an individual. These cells could then be used
for medical purposes, possibly even for growing whole organs. And
stem cells cloned from someone with a disease could be grown in
culture and studied to help researchers understand the disease and
develop treatments.
• Drug production: Farm animals such as cows, sheep, and goats are
being genetically engineered to produce drugs or proteins that are
useful in medicine.
• Used for xenotransplantation Livestock breeding and agriculture
• Production of Transgenic clones
• Production of recombinant protein such as growth hormone, insulin
through gene cloning.
• Production of live recombinant vaccines for diseases
• Detecting and monitoring diseases through gene expression and cloning
• Used in diagnosis of diseases such as viral infection (HIV), bacterial
infection (TB) –etc.
• Production of genetically modified organism.
• Cloned bacteria plays important role for the synthesis of vitamins,
hormones and antibiotics.
Limitation of cloning
• Expensive and highly inefficient.
• More than 90% of cloning attempts fail to produce a viable offspring. In addition
to low success rates, cloned animals tend to have more compromised immune
function and higher rates of infection, tumour growth, and other disorders.
• Many cloned animals have not lived long enough to generate good data about
how clones age.
• Appearing healthy at a young age unfortunately is not a good indicator of long
term survival.
• Clones have been known to die mysteriously. For example, Australia's first cloned
sheep appeared healthy and energetic on the day she died, and the results of her
autopsy failed to determine a cause of death.
Transgenic animal
A transgenic animal is one whose
genome has been altered by the transfer
of a gene or genes from another species
or breed. A foreign gene is inserted into
or deleted from the genome of the
animal to alter its DNA. This method is
done to improve the genetic traits of the
target animal.
Transgenic animals are routinely used in
the laboratory as models in biomedical
research.
Knockout Animal
• Knockout animals are a types
of transgenic animal in which
a gene is functionally inactived
by deleting or silencing it to
loss the function of this gene.
Example: Knockout mice.
Methods of creation of transgenic animals
a) DNA microinjection:
A female animal is superovulated and eggs are
collected.
The eggs are fertilized in vitro.
The transgene containing solution is injected
into the male pronucleus using a micropipette.
Eggs with the transgenes are kept overnight in
an incubator to develop to a 2 cell stage.
The eggs are then implanted into the uterus of
a pseudo - pregnant female (female which has
been mated with a vasectimized male the
previous night)
b) Embryonic stem cell-mediated gene transfer:
c) Retrovirus-mediated gene transfer:
A retrovirus genome is RNA rather than
DNA.
The code in the viral RNA is “reverse
transcribed” to produce DNA, which is
then incorporated into the host cell. For
use in transgenesis, viral RNA is modified
and the viri are introduced into an animal.
The invading viri cause the animal cells to
produce and incorporate DNA
corresponding to the viral RNA.
Transgenic animals produced using
retrovirus-mediated gene transfer are
chimeras.
Application of transgenic animals
• Study of gene regulation and their effect in physical growth and
development
• Products/characters desired in transgenic animals.
• Xenotransplantation
• Production of pharmaceuticals from Transgenic animals
• Transgenic animals as disease models in research laboratories
• Transgenic animals and disease control in human population
• Transgenic animals and Human gene therapy
• Vaccine Safety
