cloning genes into animals transgenic animal carries foreign gene inserted into its genome
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Cloning genes into animalsTransgenic animal carries foreign gene
inserted into its genome.
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Transgenic goats Ch. 10 pg. 281Produce human protein
(drug) in milk
Pharming
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Transgenic animals to produce human protein in milkMammary gland-specific promoterExample: Human EPO gene
Where is human EPO made in goat?
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Microinjection 1. Inject gene construct into animal fertilized egg, it integrates into chromosome
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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) ?
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Probed gel of goat kid DNA
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Or PCR
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3. Transgenic kid to produces human drug – how, where?
Mammary tissue specific
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4. purify drug (protein) from milk• One herd can supply the world’s need• Clean, disease free
Pail of milk with EPO
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• 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
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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?
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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
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MedicineThis chicken produces a antibody in her eggs
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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
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Fish farminggenetically engineered salmon grow faster
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PatentingRaw products of nature are not
patentable.Millions of patents Can patent a gene, a method, an animal
etc..
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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
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Reproductive cloning
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Reproductive cloningEmbryo twinning
1 sperm + 1 egg - 2 embryos (genetically identical)
http://learn.genetics.utah.edu/units/cloning/whatiscloning/
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Nuclear transfer method - The clone is a genetic copy of the donor
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SCNT = somatic cell nuclear transfer
1997 Ian Wilmut
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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
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3. Grow embryo for 6 days in lab4. Implant into surrogate mother
277 embryos -> 1 lamb (Dolly)
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Our somatic nuclei (DNA from a differentiated cell) can be reprogrammed to embryonic state!
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Cloning gamehttp://learn.genetics.utah.edu/units/cloning/cloningornot/
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Why clone animals?Models for diseasePharmingEndangered species – ex. Mouflon sheep, the
surrogate mother was a domestic sheep!Reproduce deceased petHelp infertile couples?
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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
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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
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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
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All countries have banned human reproductive cloning.
Dark brown = permissive policy light brown = flexibleYellow = no federal government funding
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The debateInterested in learning more:
2005 Lauren/diabetes/stem cells NOVA video
2006 stem cells NOVA video
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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?
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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
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Blastocyst ~ 100 cells, day 4Hollow ball of cells with inner cell mass
ICM -> embryo Blastocyst animationdevelopment in vivo
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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
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Stem cells
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QuestionsSperm?Fertilization?Embryo?
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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
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Multipotent stem cells give rise to a number of cell typesexample: stem cells in bone marrow
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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
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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
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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?
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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
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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.
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Libraries Ch 10
How to find a gene to clone If sequence is known PCR If sequence is not known library
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Genomic library = Collection of clones that contain entire genome
Need > 50,000 bacterial clones to hold the entire human genome
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Each colony contains different fragment of DNA
fragments unordered
Need many plates
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Caveats1. Restriction enzymes may cut within genes2. Need a lot of rbacteria to represent entire
genome
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cDNA library Isolate mRNA cDNACoding regions onlyTissue specific
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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
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Chromosome specific library