Cloning genes into animalsTransgenic animal carries foreign gene

inserted into its genome.

Transgenic goats Ch. 10 pg. 281Produce human protein

(drug) in milk

Pharming

Transgenic animals to produce human protein in milkMammary gland-specific promoterExample: 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 HHMIFind the 4 model organisms: mice, yeast,

fruit fly, nematode wormNote the Parkinsons mouseWhere does injected foreign DNA incorporate

into mouse genomeWhat is a pronucleus?What is done with the mouse pup tails?

AgricultureThis pig is genetically engineered to be able

to digest more and produce less manure

Other pigs produce meat high in omega 3 fatty acids

MedicineThis chicken produces a antibody in her eggs

XenotransplantationPigs have similar sized organs to humansKnock out pig cell surface antigens

to prevent hyperacute rejections

100,000 in US await organ transplantation - ~ 20,000 will get organs

Fish farminggenetically engineered salmon grow faster

PatentingRaw products of nature are not

patentable.Millions of patents Can patent a gene, a method, an animal

etc..

3 types of cloning1. gene cloning

Recombinant bacteria (as in lab)Transgenic plantsTransgenic animals

2. reproductive cloningYields an organismEmbryo twinning or nuclear transfer

3. therapeutic cloningnuclear transfer for stem cells to treat disease

Reproductive cloning

Reproductive cloningEmbryo twinning

1 sperm + 1 egg - 2 embryos (genetically identical)

http://learn.genetics.utah.edu/units/cloning/whatiscloning/

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 Go

2. Place nucleus into enucleate egg

http://learn.genetics.utah.edu/units/cloning/

Somatic cell nuclear transfer videos

3. Grow embryo for 6 days in lab4. Implant into surrogate mother

277 embryos -> 1 lamb (Dolly)

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

Cloning gamehttp://learn.genetics.utah.edu/units/cloning/cloningornot/

Why clone animals?Models for diseasePharmingEndangered species – ex. Mouflon sheep, the

surrogate mother was a domestic sheep!Reproduce deceased petHelp 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 functionEmbryo may not divideEmbryo may not implantMiscarriage

Large offspring syndrome (LOS)abnormally large organs

Abnormal gene expressionWe 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 implicationsIs 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 = flexibleYellow = no federal government funding

The debateInterested in learning more:

2005 Lauren/diabetes/stem cells NOVA video

2006 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 stage3 day embryo (morula) 5 day blastocyst

Cells at this stage are totipotent and undiffferentiated

Blastocyst ~ 100 cells, day 4Hollow ball of cells with inner cell mass

ICM -> embryo Blastocyst animationdevelopment 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

QuestionsSperm?Fertilization?Embryo?

Types of stem cellsTotipotent 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 typesexample: stem cells in bone marrow

Sources of stem cells1. Therapeutic cloning (SCNT)

Advantage = no immune rejectionNot dependent on transplant from another

person

2. Left over in vitro fertilization embryos3. Donated sperm and eggs4. 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 cells1. 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

Caveats1. Restriction enzymes may cut within genes2. Need a lot of rbacteria to represent entire

genome

cDNA library Isolate mRNA cDNACoding regions onlyTissue specific

Alcohol dehydrogenase Lane 1 RNA markerLane 2 total RNA

(Liver)Lane 3 BrainLane 4 CerebellumLane 5 CerebrumLane 6 KidneyLane 7 LiverLane 8 LungLane 9 SpleenLane 10 ThymusLane 11 Testis

51

Northern blot to assay mRNA levels in various tissues

Tissue specific expression

Chromosome specific library