Cloning involves the production of genetically identical individuals through asexual reproduction. Molecular cloning involves amplifying identical copies of DNA molecules using living organisms. The key steps of molecular cloning are fragmentation of DNA, ligation of DNA pieces in a desired sequence, transformation of cells by inserting new DNA, and selection of successfully transfected cells. Cloning has potential benefits but also risks, and human reproductive cloning remains controversial.

1. Клонирование

2. Клонирование
κλών — «веточка, побег, отпрыск»
― точное воспроизведение какого-либо объекта
― (в биологии) получение генетически
идентичных «индивидуумов»
Клон – продукт клонирования
Молекулярное клонирование - наработка
большого количества идентичных ДНК-молекул с
использованием живых организмов.

3. Recombinant DNA technology
aka DNA cloning, gene cloning, or molecular cloning
 Интересующий ген вырезается с
помощью рестриктаз или
нарабатывается в ходе ПЦР.
 Он встраивается в плазмидную
ДНК или в другой вектор.
 Векторы способны
реплицироваться в клетке.
 Экспрессия рекомбинантной ДНК
→ синтез рекомбинантных
белков.

4. Молекулярное клонирование
1. Fragmentation - breaking apart a
strand of DNA
2. Ligation - gluing together pieces
of DNA in a desired sequence
3. Transformation- inserting the
newly formed pieces of DNA into
cells
4. Screening/selection - selecting
out the cells that were
successfully transfected with the
new DNA

5. Клонирование
κλών — «веточка, побег, отпрыск»
― (в биологии) получение генетически
идентичных «индивидуумов»
Клон – продукт клонирования
организм клетка

6. Клон
― организм, являющийся генетически точной
копией другого организма
― группа генетически идентичных клеток

7. Клонирование в природе
• Бесполое размножение
• Партеногенез
• Монозиготные (однояйцевые близнецы)

8. Бесполое размножение

9. Diagram of plant cloning through
tissue culture propagation

10. Партеногенез
― одна из форм полового размножения организмов,
при которой женские половые клетки (яйцеклетки)
развиваются во взрослый организм без
оплодотворения.

11. Однояйцевые близнецы
Однояйцевые близнецы – клоны друг друга
fertilisation
Fertilised egg cell
splits
Sperm cell
Egg cell
Baby
Baby

12. History of cloning
1962 - Scientists clone frogs from blastula cells, but fail to produce tadpoles from
differentiated cells.
1962 - John Gurdon claims to have cloned frogs from adult cells.
1963 - J.B.S. Haldane coins the term 'clone.'
1966 - Establishment of the complete genetic code.
1967 - Enzyme DNA ligase isolated.
1969 - Shapiero and Beckwith isolate the first gene.
1970 - First restriction enzyme isolated.
1972 - Paul Berg creates the first recombinant DNA molecules.
1973 - Cohen and Boyer create first recombinant DNA organisms.
1977 - Karl Illmensee claims to have created mice with only one parent.
1979 - Karl Illmensee makes claim to have cloned three mice.
1983 - Kary B. Mullis develops the polymerase chain reaction technique for rapid DNA
synthesis.

13. 1983 - Solter and McGrath fuse a mouse embryo cell with an egg without a nucleus, but fail
to clone using their technique.
1985 - Steen Willadsen clones sheep from embryo cells. Steen Willadsen joins Grenad
Genetics to commercially clone cattle.
1986 - Steen Willadsen clones cattle from differentiated cells.
1986 - First, Prather, and Eyestone clone a cow from embryo cells.
1990 - Human Genome Project begins
1996 - Dolly, the first animal cloned from adult cells, born.
1997 - President Bill Clinton proposes a five year moratorium on cloning.
1997 - Richard Seed announces his plans to clone a human.
1997 - Wilmut and Campbell create Polly, a cloned sheep with an inserted human gene.
1998 - Teruhiko Wakayama creates three generations of genetically identical cloned mice.
2004 - Dr. Hwang Woo Suk claims to have cloned human embryos. His work is not able to
be replicated.
Contd……

14. First experiment to clone a frog
• 1950’s first experiments done with Frogs.
– When Transplanted nuclei from cells of Tadpoles and Frog
Embryos into egg cells that had their nuclei
removed(Briggs and King) They found that many of the
eggs would develop into tadpoles if the source of the
original nucleus was the early embryo. When they took
Tadpole nuclei they would not.
– Also found that the tadpoles that did arise would not
develop into adults
– Results gave support to the idea that differentiated cells
could not be used to create clones

15. Gurdon's experiment to clone a frog
1962

16. Cloning of mammals
• 1980th – 1990th Various mammals were
successfully cloned from embryonic cells.

17. Heres Dolly!!!
• Wilmut et al. Produced “Dolly” in 1996
• Used Genetic Information Taken from Udder
of Adult Sheep
– In order for differentiated cells to be cultured to
produce an undifferentiated embryo the process
of Cell Differentiation had to be reversed.

18. Somatic-cell nuclear transfer
Roslin Technique

19. Nuclear transfer

20. Проблемы пересадки ядер
• Как правило, проходит неудачно (требуется
много ооцитов)
• Нарушение развития эмбрионов
• Клоны – не 100% генетические копии

21. Клонирование животных
• Репродуктивное – развитие клонированного
эмбриона с целью получения полноценного
индивида
• Терапевтическое – культивирование клеток для
терапии заболеваний (получение стволовых
клеток)

22. Reproductive Cloning
 The generation of a new animal that has the same
nuclear DNA as a previously existing animal.
 Artificial Embryo Twinning: A blastomere is induced to
split, forming identical twins.
 Nuclear Somatic Transfer: The nucleus of an adult body
(somatic) cell is transferred into an egg which has had its
nucleus removed. After treatment to make it begin
dividing, the embryo is transplanted into a host uterus.
 Extremely inefficient, most eggs do not develop into an
organism.

23. Клонирование методом разделения
эмбрионов

24. Therapeutic Cloning
 Uses the process of nuclear somatic transfer to create an embryo.
 However, the embryo is destroyed and harvested for stem cells.
 An embryo must be destroyed, whether it be naturally or artificially
created.
 Stem cells are undifferentiated and retain the ability to develop into
many cell types depending on their potency.

25. Here's how therapeutic cloning works:
• DNA is extracted from a sick person.
• The DNA is then inserted into an enucleated
donor egg.
• The egg then divides like a typical fertilized egg
and forms an embryo.
• Stem cells are removed from the embryo.
• Any kind of tissue or organ can be grown from
these stem cells to treat the sick.

26. How is Therapeutic Cloning Done?
Eggs are coaxed to mature in a culture dish. Each has a remnant egg cell called
the polar body and cumulus cells from the ovary clinging to it.

27. While an egg is held still with a pipette, a needle is used to drill through the
zona pellucida, removing a plug.

28. After ejecting the zona plug, the needle is inserted back in the egg through
the hole to withdraw and discard the polar body and the egg's genetic
material.

29. A cumulus cell from another egg is taken up into the needle. Cells called
fibroblasts (or their nuclei) can also be used in this step.

30. The cumulus cell is injected deep into the egg that has been stripped of its
genetic material.

31. The injected egg is exposed to a mixture of chemicals and growth factors
designed to activate it to divide.

32. After roughly 24 hours, the activated egg begins dividing. The cells contain
genetic material only from the injected cumulus cell.

33. By the fourth or fifth day, a hollow ball of roughly 100 cells has formed. It
holds a clump of cells called the inner cell mass that contains stem cells.

34. The blastocyst is broken open, and the inner cell mass is grown in a culture
dish to yield stem cells.

35. The stem cells, in turn, can be coaxed to grow into a variety of cells that might
one day be injected into patients.

36. Стволовые клетки
Тотипотентные (омнипотентные) → любой тип клеток
Плюропотентные → практически все типы клеток, кроме внешних
эмбриональных тканей
Мультипотентные → родственные
типы клеток
Олигопотентные → несколько
типов клеток
Унипотентные → только один
тип клеток

37. Стволовые клетки
• Эмбриональные
• Соматические
• Индуцированные плюрипотентные

38. Эмбриональные стволовые клетки

39. Соматические стволовые клетки
• Гемопоэтические
• Мезенхимальные
• Кишечные
• Эндотелиальные
• Нейрональные
• и т.д.

40. Терапия стволовыми клетками

42. iPS cells
(induced pluripotent stem cells)

43. Tetraploid complementation

49. За и против
клонирования позвоночных и человека
• За
– получение животных с заданными свойствами
– восстановление вымирающих или даже вымерших
видов
– исследование и лечение многих заболеваний
– решение проблемы бесплодия
– «оживление» человека
• Против
– снижение генетического разнообразия
– низкая эффективность технологии в данный момент
– возможные нарушения развития клона
– религиозные и моральные проблемы

50. Possible Benefits of Cloning

51.  The possibility of xenotransplantation (organ
transplantation from 1 species to another) Pig
hearts, for example
 Сreation of a vast amount of new drug
development
 Disease-resistant Farm Animals
Genetically-Modified Animals

52. Infertility Patients
 Will allow infertility patients to have their own
biological child (current infertility treatments
are only about 10% effective and very costly
both monetarily and mentally on the parents)
 Will allow parents to have offspring that are
free of genetic disease (cystic fibrosis,
Huntington’s, etc…)

53. New Possibilities for Organ Transplants
 Organs, such as livers and kidneys, could be
cloned
 These clones would be more successful than
current transplants because they are created
from the patient’s body and would be free of
immune disease reactions
 Also, immuno-suppressed animals can harvest
organs for more options

54. Rejuvenation
 A human’s DNA begins to break down when
the baby is about 6 months old
 Some researchers believe that some of the
effects of aging could be reversed in the future
with the use of cloning

55. Scientific Concerns/Risks
Involved in Cloning

56. Extremely High Failure Rate
 Animal cloning has proven highly unsuccessful
 Dolly (sheep)- only 20 embryos grew out of over 277
attempts
 Snuppy- due to the highly complicated reproductive
system of dogs, the South Korean team only
obtained three pregnancies from more than 1,000
embryo transfers into 123 recipients

57. Problems During Later Development
 Out of the 20 sheep embryos that grew, 19 were
either stillborn or stopped developing due to birth
defects (Dolly was the only survivor)
 1 of the 3 puppy embryos that was growing
miscarried and 1 died shortly after birth (Snuppy was
the only survivor out of over 1,000 attempts)
 Most clones are born with Large Offspring Syndrome
(they are abnormally large) This means they have
larger organs, which leads to breathing, blood flow,
and other problems

58. Abnormal Gene Expression
 Direct comparison of gene expression profiles of more than
10,000 genes showed that for both donor cell types
approximately 4% of the expressed genes in the NT placentas
differed dramatically in expression levels from those in
controls
 This study done on mice also showed an abnormal gene
expression in the livers of cloned mice
 The clones may express different amounts of different genes
than normal humans or animals at different times

59. Telomeric Differences
 As cells divide, their chromosomes get shorter
because their telomeres shrink each time
 If the transferred nucleus is older, telomeres
could be shorter than normal in the clones
produced
 Scientists do not know the ramifications of
differences in telomeric length

60. В большинстве стран
• Разрешены эксперименты с
эмбриональными стволовыми клетками
человека
• Запрещено репродуктивное клонирование
человека

61. Создание «искусственных»
организмов

62. Синтетическая биология
• Изучение жизни путем ее построения, а не
разбора на части
• Активное использование методов генной
инженерии и клеточных технологий
• Дизайн и создание новых биологических
систем, изменение существующих

63. ГМО
― организм, генотип которого был искусственно
изменён при помощи методов генной
инженерии.
Применение
1. В исследованиях
2. В медицине, фармацевтике
3. В сельском хозяйстве
4. Развлекательное

64. Danio rerio GloFish

65. Опасность ГМО
• Аллергия
• Горизонтальный перенос генов
• Вытеснение естественных видов
ГМО как таковые не более опасны, чем, например,
традиционные технологии селекции

67. Искусственный геном
Mycoplasma mycoides

68. СПАСИБО