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.

The cloning process that produced Dolly

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