Transcript somatic cell nuclear transfer

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
 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
Reproductive cloning
Embryo twinning
 1 sperm + 1 egg - 2 embryos (genetically identical)
 http://learn.genetics.utah.edu/units/cloning/whatiscl
oning/
Nuclear transfer method - The clone is a
genetic copy of the donor
SCNT =
somatic cell
nuclear transfer
1997 Ian Wilmut
http://learn.genetics.utah.edu/units/cloning/
1. Obtain somatic cell from donor ewe
Serum starve to induce Go
2. Place nucleus into enucleate egg
 Somatic cell nuclear transfer videos
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
http://learn.genetics.utah.edu/units/cloning/clonin
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
flexible
Yellow = no federal government funding
light brown =
The debate
Interested in learning more:
2005 Lauren/diabetes/stem cells NOVA video
2006 stem cells NOVA video
Therapeutic cloning
 Obtain embryonic stem
(ES) cells

1. Isolate nucleus from a
somatic cell – which?
2. Enucleate a donor egg
How many chromosomes in
nucleus of somatic cell?
 Somatic cell nuclear transfer
 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.
~100 cells
How do we get the cells to differentiate into what we want?
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
ES cells/embryo
Therapeutic cloning
Reproductive cloning -> Implant
into female (uterus)->- birth
ILLEGAL, rarely successful in animals
Cells divide to produce more ES cells
Use to treat /cure disease
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.
2.
Restriction enzymes may cut within genes
Need a lot of rbacteria to represent entire genome
 cDNA library
 Isolate mRNA cDNA
 Coding regions only
 Tissue specific
Tissue specific expression
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
Northern blot to assay mRNA levels in various tissues
51
Chromosome specific library