Cloning genes into animals Transgenic animal carries foreign gene inserted into its genome.

Transgenic goats Ch. 10 pg. 281 Produce human protein (drug) in milk Pharming

Transgenic animals to produce human protein in milk Mammary gland-specific promoter Example: Human EPO gene Where is human EPO made in goat?

Microinjection 1. Inject gene construct into animal fertilized egg, it integrates into chromosome

2. Implant embryo into surrogate mother -> kid How do we know if kid is transgenic (has human EPO gene in its DNA from every cell) ?

Probed gel of goat kid DNA

Or PCR

3. Transgenic kid to produces human drug – how, where? Mammary tissue specific

4. purify drug (protein) from milk One herd can supply the world’s need Clean, disease free Pail of milk with EPO

Spider silk (BioSteel) –The dragline form of spider silk is the strongest material known; 5 times stronger than steel and twice as strong as Kevlar. genus Araneus

Mouse model organism HHMI Mouse model organism Find the 4 model organisms: mice, yeast, fruit fly, nematode worm Note the Parkinsons mouse Where does injected foreign DNA incorporate into mouse genome What is a pronucleus? What is done with the mouse pup tails?

Agriculture This pig is genetically engineered to be able to digest more and produce less manure Other pigs produce meat high in omega 3 fatty acids

Medicine This chicken produces a antibody in her eggs

Xenotransplantation Pigs have similar sized organs to humans Knock out pig cell surface antigens to prevent hyperacute rejections 100,000 in US await organ transplantation - ~ 20,000 will get organs

Fish farming genetically engineered salmon grow faster

Patenting Raw products of nature are not patentable. Millions of patents Can patent a gene, a method, an animal etc..

3 types of cloning 1. gene cloning Recombinant bacteria (as in lab) Transgenic plants Transgenic animals 2. reproductive cloning Yields an organism Embryo twinning or nuclear transfer 3. therapeutic cloning nuclear transfer for stem cells to treat disease

Reproductive cloning

Embryo twinning 1 sperm + 1 egg -  2 embryos (genetically identical) oning/ oning/

Nuclear transfer method - The clone is a genetic copy of the donor

SCNT = somatic cell nuclear transfer 1997 Ian Wilmut

1. Obtain somatic cell from donor ewe Serum starve to induce G o 2. Place nucleus into enucleate egg Somatic cell nuclear transfer videos Somatic cell nuclear transfer

3. Grow embryo for 6 days in lab 4. Implant into surrogate mother 277 embryos -> 1 lamb (Dolly)

Our somatic nuclei (DNA from a differentiated cell) can be reprogrammed to embryonic state!

Cloning game gornot/

Why clone animals? Models for disease Pharming Endangered species – ex. Mouflon sheep, the surrogate mother was a domestic sheep! Reproduce deceased pet Help infertile couples?

Problems with reproductive cloning High failure rate < 3% success rate 2003 first horse cloned (Prometea) 22 embryos, 800 eggs Enucleate egg may not function Embryo may not divide Embryo may not implant Miscarriage

Large offspring syndrome (LOS) abnormally large organs Abnormal gene expression We don’t understand how the nucleus is reprogrammed (its old DNA in a new egg!) Telomere problems Older DNA has shortened telomeres, some clones show lengthened telomeres

Ethical implications Is human cloning "playing with nature?" What about in vitro fertilization or hormone treatments? If a clone originated from existing person, who would be parents? Social challenges a cloned child might face Regulation

All countries have banned human reproductive cloning. Dark brown = permissive policy light brown = flexible Yellow = no federal government funding

The debate Interested in learning more: 2005 Lauren/diabetes/stem cells2005 Lauren/diabetes/stem cells NOVA video 2006 stem cells2006 stem cells NOVA video

Obtain embryonic stem (ES) cells 1. Isolate nucleus from a somatic cell – which? 2. Enucleate a donor egg Somatic cell nuclear transfer Therapeutic cloning How many chromosomes in nucleus of somatic cell?

3. inject somatic cell nucleus into enucleate egg 4. Grow to blastocyst stage 3 day embryo (morula) 5 day blastocyst Cells at this stage are totipotent and undiffferentiated

Blastocyst ~ 100 cells, day 4 Hollow ball of cells with inner cell mass ICM -> embryo Blastocyst animation development in vivo

5. Take inner cell mass, transfer to flask, and ES cells reproduce. How do we get the cells to differentiate into what we want? ~100 cells

Stem cells

Questions Sperm? Fertilization? Embryo?

Types of stem cells Totipotent stem cells (ES) can differentiate into any cell type including placenta Example: early embryo Pluripotent stem cells (ES) - 5 day embryo blastocyst cells can differentiate into any body cell type

Multipotent stem cells give rise to a number of cell types example: stem cells in bone marrow

Sources of stem cells 1. Therapeutic cloning (SCNT) Advantage = no immune rejection Not dependent on transplant from another person 2. Left over in vitro fertilization embryos 3. Donated sperm and eggs 4. Umbilical cord blood, placental blood, bone marrow

Therapeutic cloning is not reproductive cloning Reproductive cloning -> Implant into female (uterus)->-  birth ILLEGAL, rarely successful in animals Therapeutic cloning Cells divide to produce more ES cells Use to treat /cure disease ES cells/embryo

Uses of ES cells 1. tissue transplants – new liver cells, pancreas cells 2. Replace lost cells: Alzheimer disease, spinal cord injury, Parkinson’s disease, multiple sclerosis, diabetes, burned tissue, stroke, lung disease, heart disease, arthritis NOTE – ES cells cannot develop into a fetus – why?

A woman cannot have biological children and would like one. Her eggs are mixed with her husband’s sperm in a dish and a resulting embryo is implanted A woman cannot have biological children and would like one. One of her nuclei is mixed in a dish with an enucleate egg and a resulting embryo is implanted

A person with diabetes is near death due to kidney failure. Even though he took insulin throughout life, the toll of the disease on his organs is critical. He is on a waiting list for a kidney transplant. A person with diabetes uses one of his nuclei to make stem cells which are induced to form pancreatic cells in a Petri dish. His diabetes is cured at age 5.

Libraries Ch 10 How to find a gene to clone If sequence is known  PCR If sequence is not known  library

Genomic library = Collection of clones that contain entire genome Need > 50,000 bacterial clones to hold the entire human genome

Each colony contains different fragment of DNA fragments unordered Need many plates

Caveats 1. Restriction enzymes may cut within genes 2. Need a lot of rbacteria to represent entire genome

cDNA library Isolate mRNA  cDNA Coding regions only Tissue specific

Alcohol dehydrogenase Lane 1 RNA marker Lane 2 total RNA (Liver) Lane 3 Brain Lane 4 Cerebellum Lane 5 Cerebrum Lane 6 Kidney Lane 7 Liver Lane 8 Lung Lane 9 Spleen Lane 10 Thymus Lane 11 Testis 51 Northern blot to assay mRNA levels in various tissues Tissue specific expression

Chromosome specific library