Клонирование

Клонированиеκλών — «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.

Recombinant DNA technologyaka DNA cloning, gene cloning, or molecular cloning

Интересующий ген вырезается с помощью рестриктаз или нарабатывается в ходе ПЦР.

Он встраивается в плазмиднуюДНК или в другой вектор.

Векторы способны реплицироваться в клетке.

Экспрессия рекомбинантной ДНК → синтез рекомбинантныхбелков.

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

Клонированиеκλών — «веточка, побег, отпрыск»

― (в биологии) получение генетически идентичных «индивидуумов»

Клон – продукт клонирования

организм клетка

Клон

― организм, являющийся генетически точной копией другого организма

― группа генетически идентичных клеток

Клонирование в природе

• Бесполое размножение

• Партеногенез

• Монозиготные (однояйцевые близнецы)

Бесполое размножение

Diagram of plant cloning through tissue culture propagation

Партеногенез― одна из форм полового размножения организмов,

при которой женские половые клетки (яйцеклетки) развиваются во взрослый организм без оплодотворения.

Однояйцевые близнецы

Однояйцевые близнецы – клоны друг друга

fertilisation

Fertilised egg cell

splits

Sperm cell

Egg cell

Baby

Baby

History of cloning1962 - 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.

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……

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

Gurdon's experiment to clone a frog1962

Cloning of mammals

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

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.

Somatic-cell nuclear transferRoslin Technique

Nuclear transfer

Проблемы пересадки ядер

• Как правило, проходит неудачно (требуется много ооцитов)

• Нарушение развития эмбрионов

• Клоны – не 100% генетические копии

Клонирование животных

• Репродуктивное – развитие клонированногоэмбриона с целью получения полноценногоиндивида

• Терапевтическое – культивирование клеток длятерапии заболеваний (получение стволовыхклеток)

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.

Клонирование методом разделения эмбрионов

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.

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.

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.

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

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.

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.

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

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

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

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.

The blastocyst is broken open, and the inner cell mass is grown in a culture

dish to yield stem cells.

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

Стволовые клетки

Тотипотентные (омнипотентные) → любой тип клетокПлюропотентные → практически все типы клеток, кроме внешних эмбриональных тканейМультипотентные → родственныетипы клетокОлигопотентные → несколькотипов клетокУнипотентные → только одинтип клеток

Стволовые клетки

• Эмбриональные

• Соматические

• Индуцированные плюрипотентные

Эмбриональные стволовые клетки

Соматические стволовые клетки

• Гемопоэтические

• Мезенхимальные

• Кишечные

• Эндотелиальные

• Нейрональные

• и т.д.

Терапия стволовыми клетками

iPS cells(induced pluripotent stem cells)

Tetraploid complementation

За и противклонирования 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

Possible Benefits of Cloning

The possibility of xenotransplantation (organ transplantation from 1 species to another) Pig hearts, for example

Сreation of a vast amount of new drugdevelopment

Disease-resistant Farm Animals

Genetically-Modified Animals

Infertility Patients

Will allow infertility patients to have their ownbiological child (current infertility treatmentsare only about 10% effective and very costlyboth monetarily and mentally on the parents)

Will allow parents to have offspring that arefree of genetic disease (cystic fibrosis, Huntington’s, etc…)

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

Rejuvenation

A human’s DNA begins to break down whenthe baby is about 6 months old

Some researchers believe that some of theeffects of aging could be reversed in the futurewith the use of cloning

Scientific Concerns/Risks Involved in Cloning

Extremely High Failure Rate

Animal cloning has proven highly unsuccessful

Dolly (sheep)- only 20 embryos grew out of over 277attempts

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

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

Abnormal Gene Expression

Direct comparison of gene expression profiles of more than10,000 genes showed that for both donor cell typesapproximately 4% of the expressed genes in the NT placentasdiffered dramatically in expression levels from those incontrols

This study done on mice also showed an abnormal geneexpression in the livers of cloned mice

The clones may express different amounts of different genesthan normal humans or animals at different times

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

В большинстве стран

• Разрешены эксперименты с эмбриональными стволовыми клетками человека

• Запрещено репродуктивное клонирование человека

Создание «искусственных» организмов

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

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

Danio rerio GloFish

Опасность ГМО

• Аллергия

• Горизонтальный перенос генов

• Вытеснение естественных видов

ГМО как таковые не более опасны, чем, например, традиционные технологии селекции

Искусственный геномMycoplasma mycoides

СПАСИБО