ANIMAL BIOTECHNOLOGY

LESSON 12:
ANIMAL CLONING

Dear students in Today’s lecture we will discuss about the very

interesting topics Animal cloning
Objective
Introduction to animal cloning.
• To make the student understand various methods,
preparation of cloning,
• To make the student understand the animal cloning.
1. Getting the Right DNA.
2. More of the Same
3. Process
1. Getting the Right DNA
A. PCR
B. Sequencing
B. Sequenceing
i. Dideoxy 1
A. PCR ii. Dideoxy2
There are a few basic tools commonly used in the world of
molecular biology. All together and in various combinations,
they make up the bulk of our toolbox for studying genes and
gene expression.
One of these tools is the polymerase chain reaction.
When only very small amounts of a DNA to be examined are
present, we need a method to copy and produce more of the
sequence. Especially if there is a need to do this quickly, as in a
test for a disease, for example, it may take too long to prepare a
DNA fragment for ligation into a vector, cloning, and amplifica-
tion to generate enough to analyze. The polymerase chain
reaction, or PCR, is a way to amplify DNA fragments directly.
Pieces of DNA called primers that can anneal to specific
locations on a target gene are used to provide specific start sites
for DNA polymerase, an enzyme that copies DNA, to produce
a new copy of each complementary strand. These strands are
separated, and the process repeated. Each time the PCR occurs,
it results in a doubling of the DNA that is present. Repeating
the PCR 40 times results in an amplification of the target DNA ii. Dideoxy 2
sequence equal to 240 or 1,000,000,000,000 times. PCR can be
used to amplify specific regions of the DNA, or to detect
polymorphisms in the DNA similar to RFLPs. If the sequence
complementary to the primer has a mutation, then the PCR will
be Blocked, and this can be tested for. PCR can also be used to
amplify specific RNA molecules, to detect their presence. First
the RNA must be converted to a DNA molecule using reverse
transcriptase. This complementary DNA, or cDNA, molecule is
a copy of the RNA molecule that can be used for PCR, ligation
into vectors and cloning, sequencing, etc

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 a. vectors clone. We call multiple identical copies of a defined DNA
ANIMAL BIOTECHNOLOGY

b. cloning fragment, a clone. We call multiple identical copies of a genome,

a clone.
a. Vectors Types
Animal cloning then is the production of one or more identical
There are a few basic tools commonly used in the world of copies of a genome. What is a genome? All the genes carried by
molecular biology. All together and in various combinations, a cell constitute the genome. Cloning is an extension of the
they make up the bulk of our toolbox for studying genes and technology for in-vitro fertilization and embryo transfer in
gene expression. One of these tools is a vector. A vector can be common use in the agriculture industry for many years.
called a “carrier”. We use these to carry and transmit pieces of Embryo transfer, or ET, has been used since the 1950s in dairy
DNA there are several different types of vectors commonly cattle.
used in recombinant DNA work. The difference is based mainly
First, animals are caused to produce multiple ovulation through
on the size of the DNA pieces that can be carried.
the use stimulating hormones. Following mating, these
A plasmid can transmit a piece of DNA up to about 10 kilo multiple embryos are flushed out of the uterus and collected.
bases (10,000 bases) in size. Plasmids are circular pieces of DNA The embryos are then placed in the uterus of a recipient female
normally carried in bacteria. We can use the recombinant bacterial by reversing the flushing process.
plasmid to transmit DNA that we wish to study by cutting the The next stage in the technology was the development of in-
plasmid with restriction enzymes to produce sticky ends vitro fertilization, or IVF, which has been in use in cattle since
complementary to our piece of DNA and ligating them the early 1970s.
together to form a recombinant plasmid. How do we get the
In this technique, oocytes, or eggs, are removed from the ovary
bacteria to accept this foreign DNA without activating its
of the donor animal. These oocytes are taken to the laboratory
restriction defense? By using special laboratory strains where
and placed in a nutrient medium with stimulating hormones,
these defenses have been disabled.
where they will mature and become ready for fertilization.
A bacteriophage is a kind of virus that infects bacteria. The virus
The oocytes are then placed in the presence of spermatozoa,
reproduces, or replicates, itself in the bacterial host. We can
where they are fertilized. Twenty-four hours later, they begin to
insert DNA into regions of the phage DNA to produce
divide. After a few days, the developing embryos are ready for
recombinant bacteriophage that allow for replication of both
transfer into a recipient. The embryos are then placed in the
the phage and insert DNA. Bacteriophage can transmit pieces of
uterus of a recipient by reversing the flushing process.
DNA 15 to 20 kilobases in size.
A “cosmid” is yet another type of vector that can be used to The first successful cloning involved splitting embryos, first to
transmit pieces of recombinant DNA up to 40 kilobases in size. produce monozygotic twins, then triplets, quads, and so on.
An “artificial chromosome” is a vector capable of transmitting Next, it was shown that the nucleus of an embryonic cell could
even larger fragments of recombinant DNA be transferred into an oocyte from which the nucleus was
removed, and this resulted in development and eventually an
embryo that could be transferred to a recipient to establish
pregnancy.
Later, a nucleus from a fetal cell was used, and finally in 1999, a
nucleus from an adult animal cell was used.There are several
important applications of cloning in medical and health
research.
These include: -Amplification of the reproductive potential of
animals - highly regarded animals, highly prolific, productive, or
otherwise of high caliber, can be reproduced in order to have
them produce greater numbers of offspring.
Production of pharmaceuticals - transgenic animals can produce
proteins in their milk in amounts and in forms that make them
useful to treat disease - for example, animals can produce
clotting factors in milk that can be purified and used to treat
hemophilia. -Effect of genetics versus environment - if
genetically identical animals are used to test treatments for
diseases, fewer animals can be used because the effect of
b. Cloning
treatment is easier to separate from effects of animal variation
Sheep cloning animal cloning is a process to generate more than due to genetic differences.
one individual having the same genetic constitution. A clone can
be defined as one or more identical copies of the same genetic
material that is the same DNA. We use the term clone in several
different ways. We call the cells derived from a single cell by
repeated division and all having the same genetic constitution, a

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Example

ANIMAL BIOTECHNOLOGY
b. Restriction Enzymes
Restriction enzymes are used for several purposes. One of these
is to cut and then recombine pieces of DNA in particular
orders. This is what is called “recombinant DNA”. A specific
region of a gene that we wish to study can be cut out and
separated using the ability of restriction enzymes to cut at
specific sequence locations. The DNA fragment can be ligated
next to a piece of DNA from a “gene promoter” that acts as an
3. Process on/off switch. The promoter can be switched on and the gene
a. Hybridization fragment is expressed in a situation where we can test its
function alone. The DNA fragment can also be ligated into a
b. Restriction enzymes “vector”, or carrier, that allows us to increase the number of
c. Southern blot copies of the DNA so it may be more easily studied.
d. RFLP
e. GEL
a. Hybridisation
Nucleic Acids
There are a few basic tools commonly used in the world of
molecular biology. All together and in various combinations,
they make up the bulk of our toolbox for studying genes and
gene expression.One of these tools is hybridization.
DNA is normally a double-stranded molecule in which the two
strands are complementary. Because the two strands must
interact, they consist of linear orders of bases that can interact
with one another. Guanines interact with cytidines, thymines
interact with adenines; this G-C, A-T pattern is referred to as
Watson-Crick base pairing, after the two men who first
described the structure of DNA. Each strand then provides
information about the other, because they must match up, or There are a few basic tools commonly used in the world of
complement one another. molecular biology. All together and in various combinations,
Since the sequence of any single-stranded piece of DNA they make up the bulk of our toolbox for studying genes and
predicts the sequence it will be complementary to, one single- gene expression.
stranded piece can serve as the bait, or probe, for its One of these tools is a restriction enzyme. Enzymes are
complement. By placing a tag on the probe, then allowing it proteins whose function is to speed up the rate of specific kinds
seek out and anneal to its complement, we can identify specific of chemical reactions. A restriction enzyme is a class of these
DNA sequences present on a blot or in a solution. proteins produced by bacteria. Foreign DNA can be passed to
bacteria through several means, including certain viruses that
target bacteria. The bacteria’s defense against the effects of this
foreign DNA is the production of an enzyme that “restricts”
the foreign DNA by recognizing and destroying it; thus the
name “restriction enzyme”. These enzymes recognize specific
DNA sequences; different species of bacteria produce enzymes

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 that recognize different, very specific, DNA sequences. By
ANIMAL BIOTECHNOLOGY

extracting the proteins from these species of bacteria, we are able

to generate a number of different enzymes that can be used to
cut DNA in very specific ways.

Another use for restriction enzymes is in the characterization of

variation in the DNA from two individuals. If a single base in
the DNA sequence from is changed, or “mutated”, then a
restriction enzyme targeting that specific segment of the DNA
is unable to recognize the sequence and cut it. The result is a
difference in the size of the fragments generated by the enzyme.
This is called a “restriction fragment length polymorphism”, or
RFLP. This method is the basis of a number of tests for
genetic, or inherited, diseases.

c. Southern Blot
Once separated, the DNA can be looked at, or it can be trans-
ferred to a solid support, or blot. A specific labeled probe can
then be hybridized to the DNA, and this is visualized to reveal
the presence of a specific DNA sequence present in the original
sample.
When the blot is prepared from DNA separated on a gel, we call
e. GEL
that a Southern blot, named for the inventor of the method,
There are a few basic tools commonly used in the world of
Dr. Southern. When the blot is prepared from RNA separated
molecular biology. All together and in various combinations,
on a gel, we call that a northern blot, to distinguish it from the
they make up the bulk of our toolbox for studying genes and
DNA or Southern blot. The basic principle is the same for
gene expression.
both.
One of these tools is electrophoresis.
Electrophoresis is the process of separation in an electric field. It
allows molecules to be separated, or sorted, in some kind of
matrix based on how they migrate towards a positive or
negative charge. A typical matrix to sort DNA is agarose. The
agarose forms a “gel” that appears solid, but has small pores
through which molecules can travel. DNA is first loaded into
slots in the agarose gel. The buffer solution keeps the DNA
mobile allowing it to migrate though the pores in the agarose.
When the electric current is turned on, the DNA, which is
negatively charged because of its molecular structure containing
many negatively-charged phosphate groups, will move toward
the positively charged pole. The gel acts as a sieve to separate the
DNA fragments based on their size. Small pieces move through
easily, larger pieces are caught and held in the pores of the gel
matrix.
d. RFLP
I. Examination
Ii. Mapping
Examination
Examination of RFLP
Place in gel for electrophoresis, each fragment will migrate
according to its size. Shorter fragments will migrate further

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 Note

ANIMAL BIOTECHNOLOGY
Dolly was born in 1996 and revealed to the world in 1997.
Under normal circumstances, sheep can live for 10-16 years - so
she is still relatively young.
However, research suggests that Dolly may be susceptible to
premature ageing.
Dolly was created using DNA taken from an adult cell, in this
case the udder of a ewe. The fact that Dolly’s genetic material
came from a six-year-old sheep may mean that she ages quicker
than normal.
Until now, she has shown no signs of ill health and has given
birth to six healthy lambs
Current Research Work on Knimal
Cloning
Students now we knew about animal tissue culture but we need
practical oriented research work we have to now whats going in
the world
I am giving some web site address of journals
Disbelief greets claim for creation of first human clone
Alison Abbott
Description: European scientists have voiced scepticism about
claims by the Italian......
CONTEXT: ...and that Zavos was pursuing his own cloning
programme independently in Cyprus. “There is, of course,
some scepticism that the cloning has really been done,” says
Carlo Redi, an animal-cloning expert at the University of
Pavia.......
Nature 416, 570 (11 Apr 2002) News

Fetal positions
Dorothy C. Wertz
SUMMARY: The ethical opposition to cloning technologies
appears to be softening....
CONTEXT: Attitudes towards human reproductive cloning
may be quietly shifting among bioethicists. Mary Warnock, who
chaired the committee on human fertilization whose report led
to Britain’s Human Fertilisation and Embryology Act (1990),
once......
Nature 420, 269 - 270 (21 Nov 2002) Book Reviews

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