Cloning (Lecture 5) Flashcards
(24 cards)
Cloning definition
making duplicates of genetic material
Cloning in nature
- gardening
- lower animals (asexual reproduction)
- monozygotic twins
Preformationism vs. Epigenesis
- preformationism –> 1600s; came from the church; shape of embryo exists before conception from god’s vision
- epigenesis –> the shape of the embryo unfolds through development (originally from Aristotle)
Timeline to cloning
look at website with timeline
Roux-Weissman hypothesis
- 1800s
- Weissman states that genetic material diminishes with each cell division
- natural examples of regeneration made this hypothesis questionable
Hans Driesh
- 1894
- replicated sea urchins
- concluded that genetic material is not lost with cell division in vertebrates (nuclear equivalence)
- Hans Spemann replicated this in salamanders
First nuclear transplants
- 1939 Amoeba (comandom and defonbrune)
- 1952 first true vertebrate nuclear transplantation; rana pipens (briggs and king)
- 1958-1962 xenopus laevis (gurdon)
- 1962 axolotl salamander
- 1996 dolly the sheep
- 2018 hua hua and zhong zhong the monkeys
Hans Spemann early example of nuclear transfer experiment 1928
- split the one embryo into two using a piece of hair as a noose
- side with nucleus continues to divide and then undo noose and nucleus moves to other side to develop that side
- leads to two salamanders being generated
- two possibilities:
- – all cells are equal at this stage
- – cytoplasm can make the cell in effect “start over”
a fantastical experiment 1938
question: can the embryonic environment signal to somatic cell to generate animal?
test: transplant nucleus from somatic cell (non-reproductive) into egg
result: died in 1941 and then WWII so science slowed during this era
Briggs and King 1952
- blast an oocyte nucleus with radiation so it can’t divide or pluck it out
- listen to recording for rest
- concluded that somatic cells can be reverted back to a pluripotent state (aka known as SCNT)
human IVF led to …
methodology for mammalian cloning
Three varieties of cloning
- reproductive cloning (generate offspring)
- therapeutic cloning (generate stem cells for transplantation into patient)
- research cloning (in vitro modeling of diseases using patient defects and genetic background)
Research cloning
- most diseases arise from several possible genetic defects; one cell line for all will not work for modeling disease
- SCNT can be used for research cloning
Therapeutic cloning
- if the diseases allele can be corrected then the “normal” cell can be transplanted into diseased patient
- SCNT can be used for therapeutic cloning
- e.g. correcting genetic defect and then transplanting it back
Reproductive cloning - first cloned mammals
- 1984 steen willadsen performed nuclear transfer from embryos to generate cloned sheep
- 1987 neal first generated first cloned cows from embryos named fusion and copy (made clone of exact organism)
Process of cloning sheep
- donor nucleus taken from sheep’s udder
- egg cell taken from adult female sheep
- nucleus of egg cell is removed
- donor nucleus and egg cell are fused using electric shock
- fused cell divides normally
- embryo placed in uterus of a foster mother
- embryo develops normally into a lamb (dolly)
Reproductive cloning - dolly and bonnie 1997
- took so long to go from cloning frog to cloning sheep is because of size difference and other factors
- not efficient at all because so many blastocysts made were not good enough to implant, and most implanted did not develop into viable lamb (most that did develop had abnormalities)
- found that bone marrow cell was better than skin cell for outcomes
Comparing SCNT (NT-ESC) to iPSC
- pros for iPSC
- – doesn’t require egg donors
- pros for SCNT
- – have more complete reprogramming because using pluripotent environment (using egg extract instead of transcription factors)
- – can be used for mitochondrial therapy (able to replace mitochondria of donor with healthy host)
Human cloning fraud
- Hwang Woo-suk said they cloned human embryos but they didn’t
- manipulated their images
- damaged public’s outlook on stem cell research
- increased attention to research ethics of stem cell research (showed stem cell community can police itself for fraudulent activity)
Can ESCs or iPSCs generated from human cells turn into mature, functional tissues and organs?
- works in chimera mice, but not primates at blastocyst stage
- primate chimeras are possible when morula stage is used
- cloning by current methods would likely not work in humans
Human therapeutic cloning
- problem –> location of human nucleus is tough to remove
- if don’t remove host nucleus in human fibroblast, can generate blastocyst; never will be viable because triploid
Human ESCs derived by SCNT
- what hwang lied about but real deal by Mitalipov
- took so long because needed to stop development of oocyte at particular stage of mitosis
- added caffeine to change intercellular pathway
- all hallmarks of pluripotency
Monkeys cloned with dolly the sheep technique
- good for disease studies
- increased efficiency in primates by adding mrna to block deacetylace and opens up chromatin
- nothing stopping human cloning other than ethics
Mitochondrial replacement therapy
- three parent baby technique
- seems to have cured mitochondrial defect
- unfertilized donor egg with normal mitochondria and remove spindle and associated chromosomes from donated egg
- remove spindle and associated chromosomes from mother’s egg (abnormal mitochondria) and transfer into empty donor egg
- reconstituted egg is fertilized with sperm from father
- embryo with normal mitochondria and maternal and paternal genome transferred to the uterus
