7/ cloning and regeneration

Question 1

what experiment showed terminal differentiation can be reversed

Answer 1

• nuclear transfer
• human liver cell fused with mouse muscle cell
• mouse and human muscle proteins produced
• interspecies transfer allowed proteins to be easily distinguished - human myosin from mouse myosin eg

Question 2

example of damage inducing reprogramming of cells

Answer 2

• in newts and salamanders - strong regenerative capability
• iris cells de-differentiate/ transdifferentiate into lens cells to regenerate the missing tissue
• its NOT existing lens cells replacing the lens

Question 3

what conclusions can be made from nuclear transfer and iris regeneration of lens cells

Answer 3

• gene expression in nuclei from terminally differentiated cells can be changed
• gene expression can be controlled by cytoplasmic factors - yamanaka factors
• tissue loss can be sensed in some animals
• DOES NOT show terminally differentiated cells can be made totipotent

Question 4

what are clonal cells? why are they important?

Answer 4

• group of identical cells that share a common ancestry - derived from same cell
• genetically identical
• in vivo: wbcs undergo chromosomal rearrangements to generate antibodies
• in vitro: generate transgenes or mutations from cells that are identical to start off with

Question 5

first animal clones in a lab

Answer 5

frog

Question 6

frogs clones in lab 1962

Answer 6

• first done with tadpole gut cells then adult skin cells (thought to be more terminally differentiated)
• nuclei from gut/skin transplanted into unfertilised egg w/o nucleus
• gave rise to tadpoles at low rate - not adult frogs
• somatic cell nuclear transfer

Question 7

how did the researchers achieve an adult frog clone?

Answer 7

• nuclei removed from blastula
• put into unfertilised eggs w/o nuclei
• blastula nuclei far more successful - younger cells more stem cell like. as nuclei get older they lose ability to revert to stem cell
• adult frogs made - totipotency

Question 8

dolly

Answer 8

• mammary epithelial cells from donor
• induce fusion into unfertilised egg w/o nucleus using an electric current
• embryo cultured, then transferred to foster mother
• donor had a white face and host had a black face - differentiate easily. dolly white face

Question 9

stem cells in regenerative medicine

Answer 9

• focuses on in vitro
• scaffolds fabricated out of biodegradable material to support 3d growth and colonisation of cells
• stem cells from patient - avoid rejection

Question 10

stem cells in regenerative biology

Answer 10

• in vitro
• drugs or trans genes
• induce patients cells to get them to repair the damage

Question 11

create insulin producing cells - somatic cell nuclear transfer, human

Answer 11

• nucleus from skin fibroblast
• into unfertilised egg
• forms blastocyst - embryonic stem cells (ethical issues)
• cells put through differentiation protocol w growth factors and drugs
• pdx1 (regulator of pancreas differentiation) expressing cells turned into insulin producing cells

Question 12

create insulin producing cells - induced pluripotency, human

Answer 12

• skin fibroblast
• transfection (introduce modified gene into cell) with oct4, sox2, klf4
• we get iPS cells
• cells put through differentiation protocol w growth factors and drugs
• pdx1 (regulator of pancreas differentiation) expressing cells turned into insulin producing cells

Question 13

create insulin producing cells - in vivo trans differentiation, xenpous and mouse

Answer 13

• liver cells can transdifferentiate into pancreatic cells
• transfect liver cells with active pdx1 gene

Question 14

benefits of iPS cells in therapy

Answer 14

• correcting genetic defects like JEB
• replacing simple tissues or single cell types (bladder, retinal cells)
• testing how you own cells respond to different pharmaceuticals - personalised medicine. culture patients diseased cells and see how dif drugs respond

Question 15

iPS basic outline

Answer 15

• start with patient, isolate cells, yamanaka factors to dedifferentiate, other ligands to differentiate into required cell

Question 16

limitations of iPS in therapy

Answer 16

• transplantation tricky - eg deep part of brain
• organogenesis very complex - tissue engineering scaffolds have had limited success (eg heart complex organ)

Question 17

self organisation - Wilson and sponges 1907

Answer 17

• dissociated sponges by putting them through a fine sieve - the cells reorganised into an intact sponge
• adhesion molecules and cell signalling play roles

Question 18

when are organoids formed?

Answer 18

• formed by stem cells when cultured in specific media
• not just one cell type
• hope that complex tissues or organs could be grown for study and regenerative medicine
• not perfect organs, resemble simple version
• done with intestinal organoids so far - have lumen and 3 crypts, villi, lgr positive stem cells, endrocytes … etc. circular shape