Lecture 3: Stem Cells and Nuclear Equivalence

forward genetic screens (brief)

looking for genes that perform process you are interested in (discover gene underlying phenotype)

reverse genetic screens

take a known gene and mutate its sequence and observe phenotype resulting from alteration, does this match expectation? (seeing how gene sequence performs function/affects outcome)

forward vs reverse genetic screen

- forward: start with phenotype and work backward to find genotype

- reverse: start with genotype and work forward to observe resulting phenotype

getting from mutation to gene to function. how might you determine what the normal function of the mutated gene is?

catch it, break it, rescue it, move it

necessity tests

perform catch it to make inferences about what gene is doing and then break it and observe homozygous mutant to see if gene is necessary for a certain function

different tissues express

different genes

proteomics

looking at every protein in a tissue and observing the relatedness, phylogenetic tree of proteins

specific ______ activate tissue specific genes

transcription factors

Why is myosin gene expressed in muscle cells but not liver cells?

muscle cells have the MyoD transcription factor that upregulates/causes the transcription of the muscle gene, myosin, specificity of expression in muscle cells

fibroblast cells engineered to express MyoD become

muscle cells

MyoD is

sufficient to drive muscle cell differentiation in certain contexts

as development proceeds, cells become more and more

specialized, start to acquire a fate

neural ectoderm normally differentiates into

neural tissue

what happens when presumptive neural ectoderm is transplanted into early host gastrula in early gastrula stage?

it changes its fate, instead of becoming neural tissue, it becomes epidermic tissue in response to being moved to new location, implies it is getting signals and changing fate in response to new environment

what happens when presumptive neural ectoderm is transplanted into early host gastrula in late gastrula stage?

it develops based on its original fate and a neural plate tissue forms, implies it not longer responds to signals around it and differentiating according to original fate (already acquired its fate)

zygote is

totipotent

totipotent

cells that are able to develop into any type of cell found in the body

single cell zygote contains

entire genetic constitution of organism and is capable of forming all adult tissues

potency

refers to capacity of cell to produce different cell types

as differentiation proceeds, what is the relationship between potency and determination

cell fate determination increases as potency decreases

order of potency levels

toti, pluri, multi, uni

Sir John B. Gurdon experiment

- Gurdon used UV light to destroy cell nucleus in frog egg

- replaced egg nucleus with cell nucleus from differentiated intestinal epithelial cell from a tadpole

- many manipulated eggs didn't develop but several cases where normal swimming tadpoles were generated

- showed that genetic info required to generate differentiated cells in a tadpole remained intact in donor cell nucleus

- later studies showed mammals could be cloned by this technique

evidence for nuclear equivalence

move nucleus and reprogram cells

experiment studying question "are differentiated animal cells totipotent?"

- Dorset sheep 1 (donor) and Scottish blackface sheep 2

- egg cells extracted from Dorset sheep

- mammary glands (differentiated) from Scottish blackface sheep were enucleated

- nucleus of Dorset sheep cells was introduced to Scottish blackface sheep's differentiated cells through fusion process

- cell was implanted back into a Scottish blackface sheep, which gave birth to dorset sheep genetically identical to #1

what was the conclusion of the Dorset and blackface sheep experiment?

differentiated animal cells are totipotent in nuclear transplant experiments

animal vs plant totipotency

animal cells lose totipotency but plant cells retain totipotency

what can distinguish between breeds and individuals?

small differences in nucleotide sequences, which can be detected via PCR or genome sequencing

methods to detect sequence differences

1. whole genome sequencing

2. PCR

3. restriction fragment length polymorphisms (RFLP)

PCR

- sequence PCR products to identify nucleotide changes or insertions/deletions

- look at size difference of PCR products via gel electrophoresis

RFLP

- restriction fragment length polymorphisms

- use restriction enzyme which recognizes and cuts specific DNA sequence

- will cut at mutation/different allele

- use restriction enzymes to show differences in genes

RFLP was used to

show that Dolly was identical to the donor sheep

what does somatic cell nuclear transfer experiment tell us?

- no genetic information is lost as the cells pass through different developmental stages: nuclear/genomic equivalence

- cytoplasmic environment can modify a cell's fate

embryonic stem cells (ESC) are

totipotent

ESC totipotency experiment

- colony of inner cell mass from totipotent blastocysts form clumps of cells

- form colonies of embryonic stem cells

- treated with different transcription factors, causing them to form different kinds of specialized cells (heart muscle cells, pancreas islets, cartilage cells)

blastocyst

type of blastula for mammals

Shinya Yamanaka

reprogrammed cells back to embryonic state (more totipotent state)

two ways to make tissues derived from stem cells

embryonic stem cell method or induced pluripotent stem cell method

induced pluripotent stem cells experiment

- used adult fibroblast cells as template to see what factors are important in reprogramming differentiated cells

- Fbx15 is active in ESCs but not in non-ESCs, expression results in resistance to drug G418

- Fbx15 promoter drives expression of B-geo gene which encodes drug resistance

- promoter only responsive to signals in ESCs

- have 24 transcription factors (candidates), lots of combinations of them but only certain combination turned on promoter to turn on beta-geo gene and encode drug resistance (reprogrammed cells)

- four transcription factors: Yamanaka factors

- so put Yamanaka factors into cells and reprogram them to become embryonic like stem cells

- pluripotent not totipotent bc hasn't been proven to make every single cell

iPS cells

induced pluripotent stem cells contribute to normal development, can be differentiated into tissues you want or introduce them into embryos to form normal tissue

B cells and T cells lose some DNA at the immunoglobulin locus (immunoglobulin genes encode antibodies) during differentiation. What do you think would happen if you generated a mouse from an iPS cell derived from a B cell?

can't differentiate all types of cells because it is missing some DNA

organoids

little organs in petri dish that can be created from iPS cells, allow experimentation and tests on organoids rather than real human organs

pattern formation

laying down of pattern, shapes, nad regular order in a body plan

morphogen

a molecule that acts in a concentration dependent manner to induce specific developmental responses in cells