cloning and regenerative medicine Flashcards
what does Nuclear transfer show
-shows that “terminal differentiation” can be reversed
-human liver cell put into mouse muscle cell -> human liver cell and mouse muscle fused -> activation of human muscle gene expression, mouse and human muscle proteins are produced
-inter-species transfer allows the proteins to be easily analysed. In other words human myosin can be distinguished from mouse myosin
-Gene expression can be controlled by cytoplasmic factors
what can damage to cells induce
-reprogramming of cells
-if lens is removed from eye, it can regenerate from iris
-Iris cells transdifferentiate into lens cells to regenerate the missing tissue
What do we mean when cells are clonal
-A clone is a group of identical cells that share a common ancestry, meaning they are derived from the same cell.
-The implied meaning is that the cells are genetically identical.
-In vivo it is useful especially when talking about white blood cells which can undergo chromosomal rearrangement to generate antibodies.
-When culturing cells in vitro, it is important because we often generate transgenes or mutations that make cells different
-In organisms it indicates that the individual are genetically identical.
-twins= naturally occurring clones
-immune cells: genetic recombination in generating new AB
what animal was the first animal to be cloned in the lab
-frogs
-This was first done with tadpole gut cells (critisicism as gut cells aren’t seen as mature)(1962), subsequently done with adult skin cells (1975) because these are thought to be more terminally differentiated.
-Nuclei can give rise to tadpoles, but at a low rate.
-This method is sometimes called somatic cell nuclear transfer.
-Soma (body) in other words not the germline (gametes)
how was frog cloning done
-cultured adult skin cell -> removal of nucleus from cell source + UV radiation destroys DNA in nucleus of unfertilised egg -> transfer of nucleus into egg -> tadpole developed
why are younger cels more stem-cell-like
-their Blastula nuclei are far more successful
-Adult frogs can be made. This demonstrates totipotency
what was cloning the first mammal like
-donor and host (adult sheep)
-donor -> mammary epithelial cells -> culture in low serum- cells exit the mitotic cycle (G0)-> fusion induced by electric current with host
-host -> unfertilised egg -> chromosomes removed -> fusion induced by electric current with host
-fusion induced by electric current -> nucleus of mammary cell inside egg -> embryo culture -> transfer to foster mother
what are Stem cells in regenerative medicine
-Regenerative medicine focuses on using in vitro technologies to improve health
-Scaffolds are fabricated out of biodegradable materials to support 3 dimensional growth of cells.
-Stem cells should be from the patient to avoid tissue rejection
-Regenerative biology focuses upon in vivo approaches
what are the 3 strategies to create insulin producing cells
-Insulin is lacking in diabetes patients
-Insulin is normally produced in the pancreas.
-Pdx1 is a regulator of pancreas differentiation
1) skin fibroblast -> somatic cell nuclear transfer -> unfertilised egg -> blastocyst -> ES cells -> cells put through differentiation protocol with growth factors and drugs -> Pdx1- expression cells -> insulin producing glucose responsive cells
2)skin fibroblast -> induced pluripotentcy -> transfection with OCT4, SOX2 and KLF4 -> iPS cells -> cells put through differentiation protocol with growth factors and drugs -> Pdx1- expression cells -> insulin producing glucose responsive cells
3) transdifferentiation in vivo: adult exocrine pancreas liver -> transfection of organs with Pdx1 and 3 other transfection factors or with activated Pdx1 -> insulin producing glucose responsive cells
-1 +2 in humans, 3 in xenopus and mouse
what does ransfect mean
introduce a modified gene into cells or an animal (to make a transgene)
what are the advantages and lmitations of Stem cell therapiesbased upon iPS cells
-Good for
=correcting genetic defects (like junctional epidermolysis bullosa)
=replacing simple tissues or single cell types (bladder, retinal cells)
=testing how your cells will respond to different pharmaceuticals (personalised medicine)
-Limitations
=transplantation could be difficult (eg brain)
=organogenesis is very complex – tissue engineering scaffolds have had limited success
whats the initiation power of cells to self-organise
-Wilson dissociated sponges by putting them through a fine sieve then watched as the cells reorganised into intact sponges.
-Adhesion molecules and cell signalling play roles in this process.
what are organdies formed by
-stem cells when cultured in special media
-The hope is that complex tissues or organs could be grown for study and regenerative medicine
