Lecture 4: Manipulating the Genome - PDF

🥽 Lecture 4: Manipulating the genome LO: The interest in reprogramming of cells to a pluripotent state & Differentiated cells of broad origins can be reprogrammed Variability in somatic cell reprogramming is most often due to donor cell...

🥽 Lecture 4: Manipulating the genome LO: The interest in reprogramming of cells to a pluripotent state & Differentiated cells of broad origins can be reprogrammed Variability in somatic cell reprogramming is most often due to donor cell type & reprogramming method Lecture 4: Manipulating the genome 1  Many somatic cell types have been used for reprogramming! Eg) fibroblasts (mouse MEFs, human dermal fibroblasts), blood cells, renal tubular cells (urine), keratinocytes (plucked hair or skin) Examples of reprogramming factor combinations (may be cell-type specific) Yamanaka factors are still mainly used: Oct4, SOX2, Klf4, c-Myc = OSKM Thomson factors: Oct4, Sox2, Nanog, Lin28 = OSNL Omit Myc = OSK 6 factors = OSNLKM LO: Reprogramming of cells is a staged process Reprogramming is a process - cellular Lecture 4: Manipulating the genome 2  Preparing somatic cells Isolate these cells from a donor or patient Introduce reprogramming factors Introduce specific transcription factors, eg. OSKM, into the somatic cells using methods such as viral vectors Reprogramming Introduced factors (eg. OSKM) trigger genetic and epigenetic changes that reset the cell’s identity - enabling it to become pluripotent Pick clones Screen and select individual cell clones that exhibit robust pluripotency and growth characteristics Expand and characterise iPSCs Confirming the pluripotency and quality of the iPSCs Eg) checking for molecular pluripotency markers (eg. Oct4, nanog, sox2), testing for iPSCs ability to differentiate into cell types from all 3 germ layers, ensuring cells do not have significant abnormalities Lecture 4: Manipulating the genome 3  Reprogramming is a process - molecular Somatic gene expression were initially high, decreasing as reprogramming occurs; on the contrary, pluripotency-related gene expression increases over time LO: The basic advantages and disadvantages of viral, RNA, and protein-mediated reprogramming Lecture 4: Manipulating the genome 4  Viral - Lentiviral transduction, Adenovirus transduction, Sendai virus transduction Lentiviral transduction Uses lentivirus particles to deliver reprogramming factors into the target cells. Advantages: Can infect non-dividing cells Good efficiency (~0.02%) Works for many cell types LV easy to produce Disadvantages: Genomic integration can cause insertional mutagenesis Monocistronic factors (size contraints of viral packaging) Polycistronic LV Uses a single lentiviral vector carrying multiple reprogramming factors (e.g., OCT4, SOX2, KLF4, c-MYC) in a single cassette (polycistronic cassete) Advantages: Improved stoichiometry - allows for balanced expression of multiple genes, ensuring that all proteins are produced at appropriate and consistent levels Lecture 4: Manipulating the genome 5  Inducible varieties (TetO) - elements to control timing of gene expression, providing flexibility in managing when and how the reprogramming factors are expressed Disadvantages: Genomic integration Excisable polycostronic vectors: Uses vectors that include reprogramming factors in a polycistronic format with a removable selection marker. After reprogramming, the marker is excised to avoid interference. Advantages: Excisable - allows for efficient factor removal after reprogramming, reducing potential for genetic disruption. Inducible varieties Disadvantages: Labour intensive LTR gragments remain in the genome Adenovirus transduction: Delivers reprogramming factors using adenoviral vectors. The virus does not integrate into the genome, leading to transient expression. Advantages: Can infect non-dividing cells Non-integrating Disadvantages: Low efficiency Difficult to produce this virus Sendai virus transduction Utilises Sendai viruses to deliver reprogramming factors. The virus is non-integrating and does not integrate into the host genome. Advantages: ssRNA, non-integrating Lecture 4: Manipulating the genome 6  Produces high protein levels Good efficiency Non-pathogenic Disadvantages: Difficult to produce virus - commercial kits available but expensive Takes ~10 passages to dliute out viral RNA Culture at 39°C to fully remove virus Temp. sensitive vectors have weaker expression Non-viral - Piggybac transposon transfection, Episomal plasmids transfection, Minicircle vectors, miRNA transfection/infection, mRNA transfection, Protein transfection Piggybac transposon transfection Uses the PiggyBac transposon system to insert reprogramming factors into the genome. The transposons are later excised, leaving minimal genetic footprint. Advantages: Non-viral Polycistronic factors, inducible Excisable cassette - traceless Good efficiency Disadvantages: Still integrates into genome Labour intensive Excision requires a 2nd step & selection Episomal plasmids transfection Introduces plasmids carrying reprogramming factors into cells. The plasmids exist as episomes (extrachromosomal elements) that are eventually lost from the cells. Advantages: Lecture 4: Manipulating the genome 7  Non-viral Footprint-free Disadvantages: Low efficiency Some primary cell types are difficult to transfect, such as human dermal fibroblasts, requiring expensive machine Minicircle vectors Uses minicircle vectors, which are plasmid derivatives that have a minimal DNA sequence with reprogramming factors. They are non- integrating and often used for transient expression. Advantages: Non-viral, footprint-free Lower activation of exogenous silencing mechanisms compared to plasmids Disadvantages: Efficiency questionable Few cell types tested miRNA transfection / infection Advantages: Good efficiency when expressed as LV Enhance efficiencies when added with OKSM miRNA alone leave no footprint Disadvantages: Low efficiency for miR transfection alone Few published studies mRNA transfection Introduces mRNA encoding reprogramming factors into cells. The mRNA is translated into proteins without integrating into the genome. Lecture 4: Manipulating the genome 8  Advantages: Footprint-free High efficiency Disadvantages: Technically challenging to synthesise modified RNA (very fragile) Labour intensive, 7+ days of transfection Lack of published studies in non-fibroblast cell types Protein transfection Directly introduces the reprogramming proteins into cells. Advantages: Footprint-free - Immediate activity without genetic modification; avoids genomic integration. Disadvantages: Technically challenging - to produce bioactive proteins, and to get proteins to cross plasma membrane Low efficiency Few published studies in non-fibroblast cell types LO: Manipulating the genome for disease research Lecture 4: Manipulating the genome 9  Regulating expression of the genome using CRISPR/Cas9 technology Lecture 4: Manipulating the genome 10  Allows for alteration of specific site within the genome LO: Taking the next step in differentiation Pluripotency can be induced, and then induced again to differentiate into a new pathway of cell Lecture 4: Manipulating the genome 11