Induced pluripotent stem cells (iPSCs) can self-renew and differentiate into many other cell types. IPSCs are derived from somatic cells, and upon reprogramming, they share an expression profile similar to embryonic stem cells (ESCs). Among their many applications, iPSCs are an advantageous tool for disease modelling, offering an accurate system to study human molecular networks associated with specific phenotypes. Moreover, progress in genome editing technologies improved the possibilities for investigation of genotype-phenotype relationship for diseases characterized by defined genetic variants. Indeed, CRISPR/Cas9 edited iPSCs lines from healthy donors offer the possibility to investigate molecular networks with comparison to an isogenic control line. Furthermore, the ability of iPSCs to differentiate into neural cells, makes them a good model for studying neurodevelopmental diseases (NDDs). NDDs are characterized by heterogenous genetics and phenotypes. Heterozygous gene variants in the alpha 1 subunit of the sodium-voltage gated channel 1.1 (SCN1A) and in Neurochondrin (NCDN) have been associated with epilepsy. While many variants defining NDDs are associated with genes of transcriptional networks, e.g. the zinc-finger E-box binding homeobox 2 transcription factor (ZEB2) or the RPB1 subunit of RNA polymerase II complex (POLR2A). Although published animal model systems are available, there is a lack of human derived systems to investigate the gene function in disrupted molecular networks in NDDs. In this project, IPSCs deficient for SCN1A, NCDN, ZEB2 and POLR2A were generated using CRISPR/Cas9. To further evaluate the quality of the cell lines as iPSCs model, a POLR2A knock down (K.D.) line carrying a 4 bp insertion and a ZEB2 knock out (K.O.) line carrying a 790 deletion were characterized. Pluripotency and differentiation potential were confirmed by flow cytometry analysis, immunostaining, and qPCR. Both lines maintained genome integrity and editing in the top predicted off targets was excluded with PCR and Sanger sequencing screening. Furthermore, ZEB2 is involved in induction of neural crest cells (NCC); ZEB2 deficient line and the control behave similarly after a week of NCC differentiation. In contrast, POLR2A variants suggest slowing of transcription compared to the wild-type, therefore rate of transcription was measured performing an activity assay. No relevant differences between POLR2A K.D. and control line were observed in transcription rate of early pre-mRNA.
Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-445202 |
Date | January 2021 |
Creators | De Guidi, Claudia |
Publisher | Uppsala universitet, Institutionen för biologisk grundutbildning |
Source Sets | DiVA Archive at Upsalla University |
Language | English |
Detected Language | English |
Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
Format | application/pdf |
Rights | info:eu-repo/semantics/openAccess |
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