Applications in Genetics CLONING Applications in Genetics

Recombinant DNA technology (DNA cloning) Reproductive cloning 3 Main Types of Cloning Recombinant DNA technology (DNA cloning) Reproductive cloning Therapeutic cloning

Recombinant DNA technology (dates from the 1970’s) Create a bacterial plasmid containing the DNA fragment of interest Propagate the plasmid in a foreign host cell

How is the recombinant DNA created? Isolate the DNA fragment containing the gene of interest using restriction enzymes Unite it with a plasmid that has been cut with the same restriction enzymes

Besides plasmids, there are other cloning vectors: Viruses Cosmids packaged in lambda phage particles for infection into E. coli cells, BACs bacteria artificial chromosomes YACs yeast artificial chromosomes

The most common host cells for recombinant DNA are bacteria, yeast & mammalian cells

3 Main Uses of Recombinant DNA Technology Gene Therapy Genetically engineered crops & animals DNA sequencing protocols These applications are often referred to as GENETIC ENGINEERING

One of the first successful applications of genetic enginering: Commercial production of INSULIN

And then there’s the SUPERMOUSE: Put a rat growth hormone gene into freshly fertilized mouse eggs The gene was passed on to succeeding generations Over the years, transgenic mice have made a major contribution to understanding diseases such as Alzheimer's, diabetes, chronic hepatitis and cancer. 1990-U of Washington Professor Richard Palmiter and U of Pennsylvania Professor Ralph Brinster

The Benefits of Genetically Modified Crops Enhanced taste and quality Reduced maturation time Increased nutrients, yields, and stress tolerance Improved resistance to disease, pests, and herbicides New products and growing techniques Rice with increased iron and vitamins that may alleviate chronic malnutrition in Asian countries

Reproductive cloning: used to generate an animal that has the same nuclear DNA as another animal In a process called "somatic cell nuclear transfer" (SCNT), scientists transfer genetic material from the nucleus of a donor adult cell to an egg whose nucleus has been removed. See Cloning 101 http://www.dnalc.org/resources/animations/cloning101.html

The Somatic Cell Nuclear Transfer Process Step 1: Enucleation The recipient oocyte is held by a glass pipette (left side of each frame), while a glass needle is used to remove the genetic material.

The Somatic Cell Nuclear Transfer Process Step 2: Cell Transfer A cell containing the genetic material from the donor is placed inside the Zona Pellucida. An electrical pulse is then applied across the two cells, causing their membranes to fuse into one complete cell.

Nuclear Transfer The reconstructed egg containing the DNA from a donor cell must be treated with chemicals or electric current in order to stimulate cell division.

And last but not least . . . Once the cloned embryo reaches a suitable stage, it is transferred to the uterus of a female host where it continues to develop until birth. 8-cell embryo

Heeeeere’s. . . DOLLY! Born 1996 Created by Embryologist Ian Wilmut and colleagues at the Roslin Institute in Scotland Dolly the sheep was first mammal to be cloned from a differentiated cell* *From a sheep that had died years earlier

How “easy” was this experiment? Only 1 out of 277 attempts was successful!

The President reacted In response to public concern, President Bill Clinton issues a moratorium on the use of federal funds for human cloning research.

Dolly lived 6 years (1996-2003) Put down by lethal injection Feb. 14, 2003 Most Finn Dorset sheep live to be 11 to 12 years of age Dolly was a mother to six lambs, bred the old-fashioned way

Were there any problems? Possible premature aging Dolly had been suffering from lung cancer and crippling arthritis

Was Dolly a true clone? Only her chromosomal or nuclear DNA was the same as the donor Some of her genetic materials come from the mitochondria in the cytoplasm of the enucleated egg. Mitochondria contain their own short segments of DNA. Acquired mutations in mitochondrial DNA are believed to play an important role in the aging process.

Have any other mammals been cloned? Only a few . . .

7/5/98: Calves--the second adult-animal clones produced ever Ishikawa, Japan Produced by a technique similar to the one that produced Dolly The new technique would be used to breed better cattle strains with higher-quality beef or greater milk capacity Ishikawa prefectural livestock research centre

7/22/98: Three generations of cloned mice Nucleus donor Honolulu, HI 22 healthy and fertile female mice were cloned from nuclei of adult ovarian cumulus cells (ovarian cells that surround the egg and are shed with it on ovulation)  The first-born of these clone mice has been named Cumulina. 2nd generation 2nd generation

March 5, 2000: The world's first cloned piglets Produced from an adult sow using a slightly different technique from the one that produced Dolly Cloned by PPL Theraputics, Virginia USA

2000: Cloned Calves Australian gene technology researchers cloned calves

November 2001: First cloned kitten, Copycat (Cc): Created by scientists in Texas using a cell taken from an adult tortoiseshell female. Cc Rainbow

May 4, 2003: Idaho Gem, First Cloned Equine The world's first cloned mule, was born on May 4, 2003. He is an identical genetic copy of his brother, a champion racing mule called Taz.

Theraputic Cloning: the most controversial Also called "embryo cloning” Production of human embryos for use in research The goal of this process is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to treat disease.

What is a stem cell? Can be used to generate virtually any type of specialized cell in the human body: PLURIPOTENT Stem cells are extracted from the egg after it has divided for 4-5 days. The extraction process destroys the embryo, which raises a variety of ethical concerns.

Who did it first? In November 2001, scientists from Advanced Cell Technologies (ACT), a biotechnology company in Massachusetts, announced that they had cloned the first human embryos for the purpose of advancing therapeutic research. They collected eggs from women's ovaries and then removed the genetic material from these eggs with a needle A skin cell was inserted inside the enucleated egg to serve as a new nucleus. The egg began to divide after it was stimulated with a chemical called ionomycin. Although this process was carried out with eight eggs, only three began dividing, and only one was able to divide into six cells before stopping.

What medical conditions might be solved with stem cells? Heart disease Alzheimer's disease Parkinson’s disease Cancer Organ transplants

Xenotransplantation may be closer to reality than you think. . . Is there a heart, lung or kidney replacement in your future?????

Five little piggies – Dec. 25, 2001 “knock-outs”* Major step towards producing animal organs and cells for use in human transplants *Pigs lack a gene to which the human immune system reacts aggressively When an all-male litter is born and bred with the females, a true knock-out pig will be created. Noel, Angel, Star, Joy & Mary Produced by PPL Theraputics (Scotland)

ANDi, the first genetically modified rhesus monkey Cloned by embryo splitting Another step toward designing and perfecting new treatments for human genetic disorders ANDi is “inserted DNA” spelled backwards Oregon regional primate research centre in Oregon, USA

Q: What would be the value of inserting human genes suspected of causing Alzheimer disease into mice? A: Mice would serve as a model for study of this condition.

A Warning from the Roslin Institute: The scientists who cloned Dolly will stop experiments involving genetically modifying pigs for human organ transplants because of concerns that deadly new diseases could be passed on to people

South Korean scientists annouce first successful cloning of a human embryo: Thursday, Feb. 12, 2004 Used nucleus from an ovary cell and an enucleated egg from the same woman The embryonic stem cells of the resulting embryo formed muscle, bone & other tissue after 70 cell divisions. Woo Suk Hwang

But his papers were fraudulent! On May 12, 2006, Hwang was indicted on embezzlement and bioethics law violations linked to faked stem cell research. The university expelled him and the government rescinded its financial and legal support. The government has subsequently barred Hwang from conducting human cloning research.[

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

2001: Mouflon Cloned In 2001, scientists in Italy reported the successful cloning of a healthy baby mouflon, an endangered wild sheep. The cloned mouflon is living at a wildlife center in Sardinia.

What are the risks of reproductive cloning? Expensive Highly inefficient. More than 90% of cloning attempts fail to produce viable offspring. More than 100 nuclear transfer procedures could be required to produce one viable clone. More likely to have compromised immune function and higher rates of infection, tumor growth, and other disorders. Many cloned animals have not lived long enough to generate good data about how clones age.

2002 Report on Cloned Mice: Whitehead Institute for Biomedical Research in Cambridge, Mass. Genomes of cloned mice are compromised 4% of mouse liver genes function abnormally The abnormalities do not arise from mutations in the genes but from changes in the normal activation or expression of certain genes.

Imprinting is also a problem When an embryo is created from the union of a sperm and an egg, the embryo receives copies of most genes from both parents. A process called "imprinting" chemically marks the DNA from the mother and father so that only one copy of a gene (either the maternal or paternal gene) is turned on. Defects in the genetic imprint of DNA from a single donor cell may lead to some of the developmental abnormalities of cloned embryos.

Large Offspring Syndrome Pediatrics, March 2003 “. . . Even the successes come with problems. The placentas of cloned fetuses are routinely 2 or 3 times larger than normal. The offspring are usually larger than normal as well. Several months after birth one group of cloned mice weighed 72% more than mice created through normal reproduction. In many species cloned fetuses must be delivered by cesarean section because of their size. This abnormality, the reasons for which no one understands, is so common that it now has its own name--Large Offspring Syndrome."

Severino Antinori, Italian fertility specialist The Italian medical authorities warned that Dr. Antinori risked losing his right to practice in Italy because of his plans to clone human beings. March 9, 2001, Rome

Is it worth the risk?