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Stem Cells Week 5 Lecture Notes
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lMETHODS FOR GENERATING iPS CLONESImportant Considerations For the Reprogramming Method:
- What is the possible footprint?
- What is the efficiency of the reprogramming method?
- Has the method been used in different somatic cells (the one you are reprogramming)?
- Have various laboratories successfully used the method?
- Is the technique simple, or does it require specialised skills?
- What is the cost?
- Intellectual properties (for companies)
Methods for Expressing the Reprogramming Factors
Viral Transduction
- Retrovirus
- Lentivirus
- Adenovirus
- Sendai virus
Naked DNA Transfection
- mRNA
- PiggyBac
- Episomal Vectors
Retrovirus
* Any group of a RNA viruses which insert a DNA copy of
their genome into the host cell in order to replicate.
* It is unsafe for the person working with this virus. You
don’t want to produce the same virus particles and effect
the same cell.
* 3 different DNA constructs are used.
1. Presence of foreign gene. The gene that we would
like to integrate to the target cell.
2. Structural proteins, enzymes
3. Structural proteins, enzymes (envelop
protein)
The DNA’s are introduced into a cell.
RNAs are produced from the three different DNA
types. All the necessary proteins are translated.
Thus, all the building blocks to make a viral
particles are present within this packaging cell.
Viral particles then are isolated from the
packaging cell. They are transduced into the cell
line that you target.
Retroviral reprogramming only works in dividing
cells.
Takahashi Experiment: induction
of pluripotent stem cells from mouse
embryonic and adult fibroblast
cultures by defined factors.
Lentivirus
, Are a subtype of retrovirus.
They are capable of infecting non-dividing and actively dividing cell types.
Instead of using 4 constructs with the 4 reprogramming factors (multiple integration sites), researchers
started using 1 construct with all 4 (STEMCCA-STEM Cell Cassette).
Even with excisable vector there is a small
footprint retained in reprogrammed cells.
Good efficiency, easy to implement, validated for
multiple cell types.
Although the footprint is small after lentiviral
excision, non-integrative methods of
reprogramming have been developed.
The problem with all retroviral/ lentiviral
transduction is that the transgene integrates. Which
can be a problem for differentiation experiments,
and for clinical applications (oncogenes). This was
resolved (to some extent) by using the Cre/lox
system.
Cre/Lox System:
- Is a recombination system.
- It is from bacteriophage.
- After insertion of 2 LoxP sites, excision of the intervening
sequence (A) is possible after expression of the Cre
recombinase.
Adenovirus
* A group of DNA viruses discovered in adenoid tissue, most of which cause respiratory
disease.
* Contain a non-integrating linear dsDNA genome.
* Stadfeld et al. induced pluripotent stem cells generated without viral integration.
* Low efficiency, validated for only one cell type, and technically challenging.
* Has zero footprint.
Sendai Virus
* Is a non-integrating, single stranded RNA virus.
* SeV is responsible for a highly transmissible respiratory tract infection in mice,
hamsters, guinea pigs, rats and occasionally pigs, with infection passing through both
air and direct contact routes.
* NP-nucleocapsid coats the RNA.
* P-phosphoprotein has a role in replication.
* M-matrix plays a role in virus assembly.
* HN-haemoglutinin/neuroamidase,
* Glycoproteins on surface
* L-large protein functions in mRNA processing.
* Fusaki et al. efficient induction of transgene-free human pluripotent stem cells
using a vector based on Sendai virus, an RNA virus that does not integrate into the
host genome.
* Optimisation of the reprogramming factor in the Sendai virus. The expression levels of the different
reprogramming factors is important.
* Ban et al. efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by
temperature-sensitive Sendai virus vectors.
* Zero footprint. Good efficiency. Validated for multiple cell types, and reprogramming factor viral
extracts available commercially.
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