Introduction History Cell culture techniques Species cloned Approaches of cell cloning Monolayer culture- Dilution cloning Microtitration plate Suspension culture- Cloning in agar Cloning in methocel Isolation of clone By clonal rings By suspension clone Application of cell cloning Conclusion Reference

1. CELLCLONING
cell cloning
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. Synopsis-
Introduction
History
Cell culture techniques
Species cloned
Approaches of cell cloning
• Monolayer culture- Dilution cloning
Microtitration plate
• Suspension culture- Cloning in agar
Cloning in methocel
Isolation of clone
• By clonal rings
• By suspension clone
Application of cell cloning
Conclusion
Reference
cell cloning

3. Introduction
• A population of identical molecules (genes), cells or
organisms, all of which are derived from the same parent
by asexual means, is known as a clone. The process of
producing genetically similar molecules, cells, or
organisms from a common precursor by asexual
reproduction in vitro or in vivo is termed cloning.
• Cloning of continuous cell lines is much easier compared
to that of the primary cultures, and finite cell lines.
cell cloning

4. HISTORY
• 1963-J.B.S. Haldane coined the term "clone", which is
derived from the Greek word klwn, meaning "twig".
• 1978-The first baby conceived through in vitro
fertilization is born in England.
• 1994-Dr. Neal First of the University of Wisconsin clones
calves from embryos that had grown to 120 cells.
• 1996-Dolly is born at the Roslin Institute.
cell cloning

5. Cloning Techniques
• Somatic Cell Nuclear Transfer-
• In this process, the nucleus of a somatic cell is removed and
inserted into an unfertilized egg .The egg with its donated nucleus
is then nurtured and divides until it becomes an embryo. The
embryo is then placed inside a surrogate mother and develops
inside the surrogate.
• The Roslin Technique
The Roslin Technique is a variation of somatic cell nuclear transfer
that was developed by researchers at the Roslin Institute. The
researchers used this method to create Dolly.
cell cloning

6. • The Honolulu Technique
The Honolulu Technique was developed by Dr. Teruhiko
Wakayama at the University of Hawaii. In this method,
the nucleus from a somatic cell is removed and injected
into an egg that has had its nucleus removed. The egg is
bathed in a chemical solution and cultured. The
developing embryo is then implanted into a surrogate and
allowed to develop.
cell cloning

7. Dolly the Sheep
• Dolly, a Finn-Dorset ewe, was the first mammal to have been successfully cloned
from an adult cell.
• She was cloned at the Roslin Institute in Scotland and lived there from her birth in
1996 until her death in 2003 when she was six.
cell cloning

8. Species cloned
• Tadpole: (1952) Robert Briggs and Thomas J. King had successfully
cloned northern leopard frogs: thirty-five complete embryos and
twenty-seven tadpoles from one-hundred and four successful nuclear
transfers.
• Mice: (1986) A mouse was successfully cloned from an early
embryonic cell.
• Sheep: Marked the first mammal being cloned (1984) from early
embryonic cells by Steen Willadsen. Megan and Morag cloned from
differentiated embryonic cells in June 1995 and Dolly the sheep from
a somatic cell in 1996.
• Rat: Ralph, the first cloned rat (2003)
• Horse: Prometea, a Haflinger female born 28 May 2003, was the first
horse clone.
• Dog: Snuppy, a male Afghan hound was the first cloned dog (2005).
• Wolf: Snuwolf and Snuwolffy, the first two cloned female wolves
(2005)
cell cloning

9. • Water Buffalo: Samrupa was the first cloned water buffalo. It was
born on February 6, 2009, at India's Karnal National Diary Research
Institute but died five days later due to lung infection.
• Pyrenean Ibex (2009) was the first "extinct" animal (while the species
is not extinct, nor even endangered, no living examples of the
Pyrenean subspecies had been known since 2000) to be cloned back to
life; the clone lived for seven minutes before dying of lung defects.
• Camel: (2009) Injaz, is the first cloned camel.
• Pashmina goat: (2012) Noori, is the first cloned pashmina goat.
Scientists at the faculty of veterinary sciences and animal husbandry
of Sher-e-Kashmir University of Agricultural Sciences and
Technology of Kashmir successfully cloned the first Pashmina goat
(Noori) using the advanced reproductive techniques under the
leadership of Riaz Ahmad Shah.
• cell cloning

10. Cloning may be carried out in two approaches :-
• Monolayer culture:
• Petri dishes, multiwell plates or flasks can be used for
cloning by monolayer culture.
• Suspension culture:
• cloning can be carried out in suspension by seeding cells
into viscous solutions (methocel) or gels (agar).
cell cloning

11. Dilution Cloning
• Dilution Cloning Was First Introduced By Puck and Marcus, 1955
• This technique is used for the cloning of monolayer cell.
cell cloning

12. STIMULATION OF PLATING EFFICIENCY-
• Plating efficiency represents the percentage of cells
seeded at subculture that gives rise to colonies .the plating
efficiency and cloning efficiency are said to be identical ,if
each colony is derived from a single cell.
• Plating efficiency is around 10% for continuous cell lines
• For Normal Cells Plating Efficiency Drops To 0.5%-5%
• The cells at low densities require more nutrients or growth
factor.
cell cloning

13. Conditions That Improve Clonal Growth or culture factors:-
• Medium.
• Choose a rich medium, such as Ham’s F12, or a medium that has been
optimized for the cell type in use, e.g., MCDB 110 Ham
• Serum.
• When serum is required, fetal bovine is generally better than calf or
horse.
• Hormones.
• Insulin, 1 × 10−10 IU/mL, has been found to increase the plating
efficiency of several cell types
• Dexamethasone, a soluble synthetic hydrocortisone analogue,
improves the plating efficiency of chick myoblasts fibroblasts,and
melanoma and gives increased clonal growth.
cell cloning

14. Intermediary metabolites.
• Supplementing the medium with intermediary metabolites (e.g,
pyruvate, α- ketoglutarate and nucleosides stimulates plating
efficiency.
• Carbon dioxide.
• CO2 is essential for obtaining maximum cloning efficiency for most
cells. Although 5% CO2 is usually used, 2% is sufficient for many
cells and may even be slightly better for human glia and fibroblasts.
• Treatment of substrate.
• Polylysine and Fibronectin also improves the plating of many cells
• Trypsin.
• Purified trypsin used at 0.05 μg/mL
cell cloning

15. cell cloning
• Conditioned Medium-
• Medium that has been used for the growth of other cells acquires
metabolites, growth factors, and matrix products from these cells and
is known as conditioned medium.
• Feeder Layers
• A layer growth –arrested living cells , reffered to as feeder layer,
promotes plating efficiency.

16. Suspension cloning
• The transformed fibroblasts and hematopoietic stem cells,
can be more conveniently cloned in suspension rather than
monolayers
• Carried out by using agar or methocel, which can hold the
cells of a given colony together and prevent mixing of
colonies.
cell cloning

17. Suspension cloning technique in agar
cell cloning
Cells from suspension culture or
monolayer can be used
Monolayer cells have to trypsinized
while suspension cells can be
directly used
Count and dilute the cells serially
Freshly prepared agar medium with
appropriate dilution is used.
Agar medium is inoculated with the
diluted cells and incubate for 1-3
weeks.

18. Suspension cloning technique in Methocel
cell cloning

19. ISOLATION OF CLONES-
• ISOLATION OF CLONES WITH
CLONING RINGS
cell cloning

20. Why Pursue Animal Cloning Research?
• to generate groups of genetically identical animals for
research purposes
• to rapidly propagate 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
cell cloning

21. Application of cell cloning
There are many applications that nuclear transfer cloning might have
for biotechnology, livestock production, and new medical approaches.
Work with embryonic stem cells and genetic manipulation of early
embryos in animal species (including nuclear transfer) is already
providing unparalleled insights into fundamental biological processes
and promises to provide great practical benefit in terms of improved
livestock, improved means of producing pharmaceutical proteins, and
prospects for regeneration and repair of human tissues.
• In agricultural industry
• Drug production
cell cloning

22. Conclusions
• The term “clone” has many meanings but in its simplest and most
scientific sense it means the making of identical copies of molecules,
cells, tissues, and even entire animals. Research on nuclear transfer
cloning in animals may provide information that will be useful in
biotechnology, medicine, and basic science.
cell cloning

23. References
• Books : R.Ian Freshney-culture of animal cell
• U.satayanarayana-biotechnology
• Websites:
• http://www.genetics.edu.au/Information/Genetics-Fact-
Sheets/CloningandStemCellsFS26/at_dow...
• http://www.rspca.org.uk/allaboutanimals/laboratory/biotechnology/clo
nedanimals
• http://www.bioarts.com/rnd.htm
cell cloning