The document discusses cell cloning techniques, definitions, and historical milestones, including the cloning of Dolly the sheep. It outlines different methods of cloning, such as monolayer and suspension cultures, and highlights various species that have been successfully cloned. The document concludes that cloning research can advance biotechnology and medicine by aiding in disease research, organ transplants, and understanding cell differentiation.

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

2. ROADMAP
 Introduction
 What is cloning?
 Why we want to do cloning?
 History
 Technique of cell cloning
 Dolly – the sheep
 Species cloned
 Why persue animal cloning research?
 Conclusion
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3. Introduction
 Clones are organisms that are exact
genetic copies.
 Cloning is to duplicate a cell or an
organism, usually asexually which is
genetically an exact replica of the other
cell or organism.
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4. What is cloning?
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 The process of making identical genomic
copies of an organism.
 Production of one or more individual
plants or animals that are genetically
identical.

5. Why we want to do cloning?
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 Researchers hope that these techniques can be
used in researching and treating human disease
and genetically altering animals for the
production of human transplant organs .

6. History
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 1963 – J.B.S Haldane coined the term clone which is
derived from the greek word klwn meaning twig.
 1978 – 1st baby conceived through invitro fertilization is
born in england.
 1994 – Dr. Neal of wisconsin university clone calves from
embryos that had grown to 120 cells.
 1996 – Dolly is born at roslin institute.

7. Technique of cell cloning
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 There are two approaches -:
1) Monolayer culture – A type of culture in which cells are
grown in a single layer on a flask or petri dish containing
the culture medium.
2) Suspension culture – It is a type of culture in which
single cells or small aggregates of cells multiply while
suspended in agitated liquid medium.

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 Dilution cloning – It is the most commonly used
technique for cloning of monolayer cells and involves
following stages.
1) Trypsinization of cells to produce single cell suspension.
2) Dilution of cells to about 10-100 cells/ml.
3) Seed the cells in multiwell dishes, petriplates or plastic
bottles.
4) Incubate under appropriate conditions for 1-3 weeks.
5) Isolate individual colonies.

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 Suspension cloning - The technique of cloning in agar
suspension is carried out in the following stages:-
1) Cells from suspension culture or monolayers can be
used.
2) Count the cells and dilute serially so that 10-200
cells/ml are finally present.
3) Freshly prepared agar medium with appropriate
dilution is used.
4) The agar medium is inoculated with the diluted cells.
5) Incubation for 1-3 weeks give clones in culture.

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12. Dolly – The Sheep
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 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.

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14. Species cloned
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 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.

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 Dog: Snuppy, a male Afghan hound was the first cloned dog
(2005).
 Wolf: Snuwolf and Snuwolffy, the first two cloned female wolves
(2005)
 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.

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 Camel: (2009) Injaz, is the first cloned camel.
 Brahman – In February 2011, Brazil cloned.
 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.
 Cirb Gaurav – In January 2016, the scientist at the central
institute for research on buffaloes in Hisar, India announced that
they had cloned a buffalo offspring “Cirb Gaurav” using cells of
the ventral side of the tail of superior buffalo.

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 Crab eating macaque – Zhong Zhong and Hua Hua first
successful cloning of a primates using somatic cell nuclear
transfer, the same method as dolly, with the birth of two
live female clones. Conducted in china in 2017 but
reported in January 2018.

18. Why Pursue Animal Cloning Research?
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 To help scientist to identify & isolate genes in order to
understand more about their function.
 To provide research models of human disease to help
develop new drugs & new strategies for repairing
defective genes.
 To provide organs & tissue for use in human transplant
surgery.
 To produce milk which contains therapeutic protein or
to alter the composition of the milk to improve its
nutritional value for human infants.
 To pursue basic knowledge about cell differentiation.

19. Conclusion
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 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.

20. References
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 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/bi
otechnology/clonedanimals
 http://www.bioarts.com/rnd.htm