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Cloning
1. It refers to creating a genetically identical population
2. DNA can be combined by using r.e that create sticky ends in
the DNA. This rDNA has a target DNA sequence of interest
3. The target DNA sequence is carried in some type of vector,
usually a bacterial plasmid or a virus
4. The target DNA sequence is inserted into a host organism &
the natural doubling time of the organism is used to create
many copies of the target DNA sequence
5. Organisms that are carrying the target DNA are identified
through a process called selection, which often involves
antibiotic resistance.
History of cloning
1952 Northern leopard frogs cloned.
1953 Structure of DNA discovered.
History of cloning
1978 Louise, the first child
conceived through in vitro fertilization,
was born.
1993 Human embryos were first
cloned
July 5, 1996 Dolly was born.
Photo from: www.cnn.com
Dolly – the first mammal cloned using mature cell Dolly the Lamb in
1996
Method: Nuclear transfer
Organization: Roslin Institute at UK
Photo from Ming Pao 18th August 2002
Dolly
1996-2003
p.331
In July, 2016, four
identical clones of Dolly
(Daisy, Debbie, Dianna
and Denise) were alive
and healthy at nine years
old.
Two methods of cloning Embryo cloning - remove a cell from an
embryo for developing into a separate embryo.
Adult cell cloning - replace DNA/nucleus
from a cell by another.
Embryo cloning – do not know the
characteristics of the offspring.
Adult cell cloning – characteristics are almost
the same as the nucleus donor.
Advantages of animal cloning Can produce animal with a desired trait, for
Protein products, organs
Proliferate endangered animals
Cloning of woolly Mammoth
Extinct 10,000 years ago
Is there any intact cell left?
Cloning of transgenic animal Cloning of a cow
containing mad
cow disease
resistant gene
In Shangdong,
China
From Mingpao 28/4/2006
Concerns in animal cloning
Technology complicated
Survival rate of cloned embryos
low
Overweighing of calves at birth
Breeders may want to keep their
animal unique
Breeders may want to create
better offspring
Specific steps of cloning
A. The selection of a vector
B. Generation of foreign DNA fragments (containing
particular gene) and ligate the foreign DNA into vector to
make insert-vector recombinant.
C. The selection of appropriate (competent) E. coli strain
D. Transforming competent E. coli with recombinant vector
E. Grow E. coli colonies to replicate recombinant vectors and
select colonies containing the recombinants DNA.
F. Characterizing the properties of DNA inserts by genetic
analysis.
A. Cloning Vectors
Cloning vector is a plasmid that can be modified to carry new genes, which must have:
An origin of replication.
A selectable marker (antibiotic resistance gene, such as ampicillin resistance( ampr) or tetracycline resistance( tetr).
Multiple cloning site (MCS) a site where insertion of foreign DNA will not disrupt replication or inactivate essential markers.
Plasmid pBR322
Fig. 13-10, p.339
B. Generation and Ligation of Inserts into
Vector
The standard procedure for creating the
recombinant molecule involves cleaving the
DNA of interest [the insert] and the vector with
the same restriction enzyme, followed by
incubation with DNA ligase to ligate the insert
into the vector.
DNA Library
1. It is a collection of clones of an entire genome
2. The genome is digested with r.e & the pieces are
cloned into vectors & transformed into cell lines
3. Specific radioactive probes to a sequence of
interest are reacted to filters that have copies of
the bacterial colonies in the library. The probe
binds to the sequence of interest, and the colony’s
location can be seen by autoradiography
4. A cDNA library is constructed by using reverse
transcriptase to make DNA from mRNA in a cell.
This cDNA is then used to construct a library
similar to a genomic DNA library.
Fig. 13-21, p.352
Formation of
cDNA
Fig. 13-19, p.350
Steps involved in
the construction of
a DNA library
C. Choice of an E. coli host
Usually an E. coli mutant called lacZΔM15 is used as a host for the recombinant vector. This mutant is characterized by having inactive β-galactosidase activity due to deletion in the N-terminal part of the enzyme protein coded by defective lacZ gene.
In the mean time , the defective part of this lacZ gene has been inserted into the vector(plasmid DNA).So, when the plasmid vector is transferred the bacterial host the combined parts complement to each others to give the active enzyme. A test for the formation of active enzyme can be detected by converting a blue colored X-gal dye into white color.
D. Transformation of E. coli with
Recombinant Vector
Transformation is the process of making the
bacteria to take up the recombinant vector
molecule. Nucleic acids do not enter
bacteria under their own power but usually
they require certain procedure involving
treating the bacteria with ice-cold solutions
of CaCl2 followed by short heating to 42
Fig. 13-13, p.341
Clone selection
via blue/white
screening
Inclusion of ampicillin in the agar allows only
bacteria with a plasmid to grow, because these
plasmids provide antibiotic resistance
Three types of colonies are
produced:
1. Transformed bacteria containing
recombinant plasmid
2. Transformed bacteria containing non-
recombinant plasmid
3. Non-transformed bacteria
Identification of cells containing
plasmids
Inclusion of X-gal allows for blue-white colony
screening. Recombinant clones will have white
colonies while non-recombinant will have blue
colonies.
The cloning of a virus
Fig. 13-6, p.336
Fig. 13-7, p.336
The cloning of cells
Fig. 13-8, p.336
The cloning of
human DNA
fragments with
a viral vector
For A Successful Experiment
When bacteria take up a plasmid, we say they have
been transformed
Bacteria are encouraged to take up foreign DNA by:
1.Heat-shock the bacteria at 42 C. followed by placing
them on ice.
2. Place them in an electric field “electroporation”
Then selection through selectable markers on the
plasmid.
Fig. 13-12, p.340
Cloning with pU
plasmids
Cloning Human
Cloning – two kinds
Reproductive cloning – an embryo
is created and implanted into a
woman’s womb to bring it to term.
Therapeutic cloning – an embryo
is created in order to obtain cells
from it.
Why clone human?
Just an ‘unconventional’ means of
reproduction
In vitro fertilization
Surrogate mother
Adoption
Study human development
Produce spare parts
Test for genetic defect
Increase chance of pregnancy
Produce two children at the
same time
Why clone human?
Preserve traits and talents
Extension of life in unusual
circumstances
One spouse sterile
Why clone human?
Cloned embryos provide :
Brain cells for disorders like Parkinson and
Alzheimer’s disease
Pancreatic islet cells for diabetes
Nerve cells for spinal cord damage
Blood and bone marrow cells for blood cell
disorder
Positive points of therapeutic cloning
http://dels.nas.edu/bls/stemcells/types-of-stem-cells.shtml
Embryonic stem cells Adult stem cells
The use of stem cells to generate clones
1. Creating an embryo
through in vitro
fertilization,
2. culturing ES cells derived
from it to provide a
sufficient population for
the tricky task of inserting
genes
3. extracting the nucleus of a
successfully altered cell to
construct a cloned embryo
4. The resulting offspring
would have developed from
a cell derived from an
embryo created with an egg
and a sperm, and
"improved" in the
laboratory.
Why not perform reproductive cloning?
Eugenic – to maximize certain traits intentionally
Reduce genetic diversity
Use as substitute for organ
Clone may have reduced life expectancy
Clone may be abnormal
Human Reproductive Technology Bill
(2000)
Not allow the followings:
Replace nucleus of
an embryo with
nucleus of another
cell
Clone an embryo
Trading of embryo
Human cloning in China (Oct. 1, 2003) – by Ministry of Health
Prohibits surrogate mother
Prohibits reproductive human cloning
Prohibits donation of embryos
Prohibits trading of eggs
Recent Development in Human Cloning
Korean Scientists led by Dr. Woo-suk Hwang
produced cloned human embryos (Science, Feb.
12, 2004) – later found to be fabricated
American scientist Panayiotis Zavos claimed to
have cloned 14 human embryos and transferred
11 of them into the wombs of four women (April,
2009)
Recent Development in Human Cloning
August 2004 - UK granted the first licence for
work toward therapeutic cloning.
Nov. 2004 - Californians passed a $3 billion
measure to create an Institute for Regenerative
Medicine based on embryonic stem cell
research.
Please consider ...... Would the views of animal and human
cloning differ among people with different religious believes?
Is embryo a living human?
Since the use of stem cells for therapeutic cloning is still in experimental stage, would the use of cells from embryo be acceptable?
How about using the embryos left over after in vitro fertilization?
Please consider ...... Is reproductive cloning a violation of
natural birth?
How about the cloning of a beloved one
who dies accidentally?
How about cloning for sterile couples?
Under what circumstances do you want to
make a copy of yourself ?
End
