Stem Cells Flashcards
What is the definition of a stem cell?
A stem cell is a cell that has retained its capacity to self renew. It is a single cell that can replicate itself, or differentiate into many cell types.
Name the two types of stem cells
- Embryonic Stem cells
2. Adult stem cells (Somatic stem cells)
What do embryonic stem cells differentiate into?
Can differentiate into almost any cell type in the body.
What can somatic stem cells differentiate into?
Somatic stem cells reside in specific tissues and can differentiate into a subset of related cell types.
What does pluripotent mean?
Condition to be able to generate all different cell types for an adult body.
What does multipotent mean?
Multipotent is used to describe a stem cell that can make multiple different cell types but NOT ALL.
Describe the steps for isolating ES (embryonic stem cells)?
- Sperm and egg join
- Embryo develops for 5-7 days
- Remove inner cell mass from blastocyst
- Grow in Petri dish
- Change culture conditions to stimulate cells to differentiate into a variety of cell types such as skeletal muscle cells, skin cells or neural cells.
What does homologous mean?
Similar in position structure and evolutionary origin but not necessarily in function.
Describe Steps 1 and 2 for gene targeting in ES cells.
1. ES cell culture Embryonic stem (ES) cells are cultivated from mouse pre-implantation embryos- blastocysts. 2. Construction of targeting vector: the vector contains pieces of DNA that are homologous to target gene, as well as inserted DNA which changes the target gene and allows for positive/negative selection.
Describe how neo resistance works in construction of a target vector, in regards to positive/negative selection…
Within the homologous DNA we’re using as vector, we’ve constructed neo resistance, this confers resistance to an antibiotic type drug, so any cell expressing neo^r gene will be resistant to this antibiotic. This gives the possibility to positively select for cells that have taken up this DNA.
What does transfection mean?
It’s a process by which we introduce new genetic material to cells.
What is HSV-tv used for?
Negative selection
Describe step 3 in gene targeting in ES cells
The cellular machinery for homologous recombination allows the targeting vector to find and recombine with the target gene. The neomycin cassette in middle will loop out to allow this alignment. Due to high level of homologous recombination in mice ES cells recombination events will happen in some cells which will replace the original target sequence with the new sequence which now carries neo^r gene.
Describe how HSV-tk selection works…
HSV-tk carries a means of making cells susceptible to a certain treatment which kills them, cells that have made a poor alignment therefore die as they carry this, HSV to not included in cells with a good alignment.
Describe step 4 for Gene targeting in ES cells
Proliferation of targeted ES cells: selection for presence of neo^r and absence if HSV-tk enriches targeted ES cells- get a pure population of ES cells carrying targeted gene.
Describe step 5 of gene targeting in mice…
- Injection of ES cells into blastocysts
The targeted ES cells are injected into blastocysts, where they mix and form a mosaic with the cells from the inner cell mass from which the embryo develops.
The injected blastocysts are implanted into a surrogate mother where they develop into mosaic embryos.
Describe step 6 for gene targeting in mice
6. Birth and breeding of mosaic mice Mosaic mice (chimeric mice) are born and are mated with normal mice to produce both gene targeted and normal offspring.
Gene targeted mice are also called what?
Knockout mice
How many generations would it take to get a stable line of genetically altered mice?
2, the 2nd generation would produce homozygous knockouts.
Define Induced Pluripotent stem cells(iPS)
iPS are a type of pluripotent stem cells derived from adult somatic cells that have been genetically reprogrammed to an embryonic stem cell like state through the forced expression of genes and factors important for maintaining the defining properties of ES cells.
What do ES cells have that regulate the stem cells and what are the core ones called?
A set of transcription factors and the core TF’s that maintain the ES cell state are called OSKM’s
What does OSKM stand for/mean?
They are the master gene regulator proteins- Oct4, SOX2 and KLF4 (OSK) and Myc
What do OSK do?
OSK induce both their own and each other synthesis!
Why are iPS cells a promising source of cells for treating diseases?
They can be made from patients own cells so there is no risk of rejection.
