GENE CLONINGGENE CLONING

CloningCloning

Different processes for duplicating Different processes for duplicating biological material. biological material.

Diff types = technologies can be used Diff types = technologies can be used for other purposes besides producing for other purposes besides producing the genetic twin of another organism. the genetic twin of another organism.

1. DNA cloning, 1. DNA cloning, 2. Reproductive cloning2. Reproductive cloning3. Therapeutic cloning3. Therapeutic cloning. .

Recombinant DNA Technology or DNA Cloning

1970s = common practice in MB labs today1970s = common practice in MB labs today ““Recombinant DNA technology," "DNA Recombinant DNA technology," "DNA

cloning," "molecular cloning“ or "gene cloning," "molecular cloning“ or "gene cloning”cloning”

Transfer of a DNA fragment of interest Transfer of a DNA fragment of interest from one organism to a self-replicating from one organism to a self-replicating genetic element eg. bacterial plasmid. genetic element eg. bacterial plasmid.

The DNA of interest can then be The DNA of interest can then be propagated in a foreign host cell. propagated in a foreign host cell.

Scientists studying a particular gene often Scientists studying a particular gene often use bacterial plasmids to generate multiple use bacterial plasmids to generate multiple copies of the same gene. copies of the same gene.

Plasmids Self-replicating circular DNA Self-replicating circular DNA

moleculesmolecules HGP = Plasmids and other HGP = Plasmids and other

types of cloning vectors are types of cloning vectors are used by researchers to copy used by researchers to copy genes to generate sufficient genes to generate sufficient identical material for further identical material for further study. study.

Can carry up to 20,000 bp of Can carry up to 20,000 bp of foreign DNA foreign DNA

How? DNA fragment containing DNA fragment containing

the gene of interest is cut the gene of interest is cut from chromosomal DNA from chromosomal DNA using RE.using RE.

United with a plasmid that United with a plasmid that has been cut with the has been cut with the same RE. same RE.

Fragment of chromosomal Fragment of chromosomal DNA + cloning vector = DNA + cloning vector = "recombinant DNA "recombinant DNA molecule." molecule."

The recombinant DNA can The recombinant DNA can then be reproduced along then be reproduced along with the host cell DNA. with the host cell DNA.

Other cloning vectors

VirusesVirusesBACs = 100 – 300 kbBACs = 100 – 300 kbYACs. = 1 MBYACs. = 1 MBCosmids = up to 45 kbCosmids = up to 45 kb

Reproductive cloning Technology used to generate an animal that has Technology used to generate an animal that has

the same nuclear DNA as another currently or the same nuclear DNA as another currently or previously existing animal. previously existing animal.

Dolly. Roslin Institute. Dolly. Roslin Institute. Somatic cell nuclear transfer (SCNT),Somatic cell nuclear transfer (SCNT), = transfer = transfer

of GM from the nucleus of a donor adult cell to of GM from the nucleus of a donor adult cell to an egg whose nucleus, and GM has been an egg whose nucleus, and GM has been removed. removed.

The reconstructed egg containing the DNA from a The reconstructed egg containing the DNA from a donor cell must be treated with chemicals/ donor cell must be treated with chemicals/ electric current to stimulate cell division. electric current to stimulate cell division.

Once the cloned embryo reaches a suitable stage, Once the cloned embryo reaches a suitable stage, it is transferred to the uterus of a female host it is transferred to the uterus of a female host where it continues to develop until birth.where it continues to develop until birth.

Dolly the sheepDolly the sheep

The first mammal to be cloned The first mammal to be cloned was put down by lethal was put down by lethal injection Feb. 14, 2003. injection Feb. 14, 2003.

Prior to her death, Dolly had Prior to her death, Dolly had been suffering from lung been suffering from lung cancer and arthritis.cancer and arthritis.

Studies showed that telomeres Studies showed that telomeres are shortened, a phenomenon are shortened, a phenomenon that is associated with cellular that is associated with cellular aging aging

Reproductive cloningReproductive cloning 1952 = tadpole.1952 = tadpole. The possibility of human cloning, raised when The possibility of human cloning, raised when

Scottish scientists at Roslin Institute created Dolly Scottish scientists at Roslin Institute created Dolly ((NatureNature 385, 385, 810-13, 1997), 810-13, 1997),

Since Dolly, sheep, goats, cows, mice, pigs, cats, Since Dolly, sheep, goats, cows, mice, pigs, cats, and rabbits all using nuclear transfer technology. and rabbits all using nuclear transfer technology.

Attempts at cloning certain species such as Attempts at cloning certain species such as monkeys, chickens, horses, and dogs, have been monkeys, chickens, horses, and dogs, have been unsuccessful as some speciesunsuccessful as some species

? some may be more resistant to somatic cell ? some may be more resistant to somatic cell nuclear transfer than others. nuclear transfer than others.

Process can be traumaticProcess can be traumatic

Therapeutic cloningTherapeutic cloning Also called "embryo cloning," production Also called "embryo cloning," production

of human embryos for use in research. of human embryos for use in research. Not to create cloned human beings, but to Not to create cloned human beings, but to

harvest stem cells that can be used to harvest stem cells that can be used to study human development and to treat study human development and to treat disease. disease.

Stem cells are extracted from the egg Stem cells are extracted from the egg after it has divided for 5 days.after it has divided for 5 days.

Many researchers hope that one day stem Many researchers hope that one day stem cells can be used to serve as replacement cells can be used to serve as replacement cells to treat heart disease, Alzheimer's, cells to treat heart disease, Alzheimer's, CA and other diseases CA and other diseases

How to utilize recombinant tech?

Gene therapy = Tx genetic conds by Gene therapy = Tx genetic conds by introducing virus vectors that carry introducing virus vectors that carry corrected copies of faulty genes into corrected copies of faulty genes into the cells of a host organismthe cells of a host organism

GMO/GAO = Genes from different GMO/GAO = Genes from different organisms that improve taste organisms that improve taste nutritional value or provide nutritional value or provide resistance resistance

Sequencing genomes = Knowledge.Sequencing genomes = Knowledge.

How to utilize repro cloningHow to utilize repro cloning Can be used to develop Can be used to develop efficient ways to

reproduce animals with special qualities. Eg. . Eg. Mass production of , drug-producing animals or Mass production of , drug-producing animals or animals that have been genetically altered to animals that have been genetically altered to serve as models for studying human disease. serve as models for studying human disease.

Could be used to Could be used to repopulate endangered animals or animals that are difficult to breed. Eg. In 2001, that are difficult to breed. Eg. In 2001, the first clone of an endangered wild animal was the first clone of an endangered wild animal was born, a wild ox called a gaur. The young gaur died born, a wild ox called a gaur. The young gaur died from an infection about 48 hours after its birth. from an infection about 48 hours after its birth.

In 2001, scientists in Italy reported the successful In 2001, scientists in Italy reported the successful cloning of a healthy baby endangered wild sheep, cloning of a healthy baby endangered wild sheep, now living in Sardinia. now living in Sardinia.

How to utilize therapeu. How to utilize therapeu. cloning?cloning?

. . May produce May produce whole organs/ healthy cells that can replace damaged cells.that can replace damaged cells.

Eg transplants = stem cells would be used Eg transplants = stem cells would be used to generate an organ or tissue that is a to generate an organ or tissue that is a genetic match to the recipient. In theory, genetic match to the recipient. In theory, the cloned organ could then be the cloned organ could then be transplanted into the patient without the transplanted into the patient without the risk of tissue rejection. risk of tissue rejection.

If organs could be generated from cloned If organs could be generated from cloned human embryos = the need for organ human embryos = the need for organ donation could be reduced. donation could be reduced.

Problems of repro cloningProblems of repro cloning Reproductive cloning is expensive and highly inefficient. Reproductive cloning is expensive and highly inefficient. > 90% of cloning attempts fail to produce viable offspring.> 90% of cloning attempts fail to produce viable offspring. > 100 nuclear transfer procedures could be required to > 100 nuclear transfer procedures could be required to

produce one viable clone.produce one viable clone. Cloned animals tend to have compromised immune Cloned animals tend to have compromised immune

function and higher rates of infection, tumor growth, and function and higher rates of infection, tumor growth, and other disorders.other disorders.

Japanese studies have shown that cloned mice live in poor Japanese studies have shown that cloned mice live in poor health and die early. health and die early.

1/3 calves born alive have died young, and many of them 1/3 calves born alive have died young, and many of them were abnormally large. were abnormally large.

Don’t live long enough to generate good data.Don’t live long enough to generate good data. In 2002, researchers at Cambridge reported that the genomes of In 2002, researchers at Cambridge reported that the genomes of

cloned mice are compromised = certain of genes function cloned mice are compromised = certain of genes function abnormally.abnormally.

Human cloning: the most controversial debate of the

decade. 

Aroused worldwide interest and concernAroused worldwide interest and concernbecause of its scientific and ethical because of its scientific and ethical

implicationsimplications

Is it morally acceptable?Is it morally acceptable?

Certain questions to be raised

Is self-engineering acceptable? Is self-engineering acceptable? Will failures, eg. deformed offspring, Will failures, eg. deformed offspring,

be acceptable? be acceptable? Will cloning lead to designer babies ? Will cloning lead to designer babies ? Who is socially responsible for cloned Who is socially responsible for cloned

humans? humans? Rights and legal protection?Rights and legal protection?

95% public say no to cloning, 95% public say no to cloning,

but 95% of scientists say yesbut 95% of scientists say yes

Reasons for cloningReasons for cloning

Provide valuable research Provide valuable research Answer to infertility? Answer to infertility?

Scenario 1Scenario 1

Scenario 1: A husband and wife who Scenario 1: A husband and wife who wish to have children but both are wish to have children but both are carriers of a lethal recessive gene.carriers of a lethal recessive gene.

“ “ Rather than risk the one in four chance of conceiving a child who will suffer a short and painful existence”

Scenario 2Scenario 2

Scenario 2 : Parents of a terminally ill Scenario 2 : Parents of a terminally ill child are told that only a BMT can child are told that only a BMT can save the child's life:save the child's life:

“ “ With no donor available, parents attempt to clone a human being from cells of the dying child. If successful, the new child could be a match for

BMT”

Concerns of human cloningConcerns of human cloning

Act of human arrogance = Playing GodAct of human arrogance = Playing God Safety concernsSafety concerns = Due to the inefficiency = Due to the inefficiency

of animal cloning (1-2 viable offspring for of animal cloning (1-2 viable offspring for every 100 experiments) and the lack of every 100 experiments) and the lack of understanding about reproductive cloning understanding about reproductive cloning = unethical to attempt to clone humans.= unethical to attempt to clone humans.

Technology only in animals, in men = Technology only in animals, in men = possibility of mutation/ bio damage? possibility of mutation/ bio damage?

Individuality and Uniqueness =Individuality and Uniqueness = Fear Fear that a clone would not be an that a clone would not be an “individual” but merely a carbon “individual” but merely a carbon copy.copy.

Would it have a soul?Would it have a soul?Clone would be constantly compared Clone would be constantly compared

= burdened with oppressive = burdened with oppressive expectationsexpectations..

????????????????????????????????????????????????????????????????

THE END OF ML429THE END OF ML429

STUDY HARD, PRAY HARDSTUDY HARD, PRAY HARD

I WISH YOU ALL THE VERY BEST IN I WISH YOU ALL THE VERY BEST IN YOUR EXAMSYOUR EXAMS