Generation of TDGF1-knockout in a GBM cell line using CRISPR/Cas9
Author
Justesen, Daniel Lamdahl
Term
4. term
Publication year
2019
Submitted on
2019-05-31
Pages
74
Abstract
Glioblastoma multiforme (GBM) is an aggressive brain tumor with a poor prognosis. TDGF1 (Cripto‑1) regulates stem cell behavior during embryonic development and is frequently upregulated in cancers, where it can promote cell migration and invasiveness. This project aimed to generate a TDGF1 knockout in the U87 GBM cell line using CRISPR/Cas9 while evaluating cloning and transfection workflows. Four single‑guide RNAs (sgRNAs) targeting exon 3 of TDGF1 were designed (CrispOR), annealed, and cloned into PX461 plasmids via BbsI restriction cloning. Following E. coli transformation, plasmids were sequenced to confirm correct insertion. U87 cells were transfected with the cloned plasmids using either Lipofectamine or calcium phosphate, GFP‑positive cells were isolated by FACS, and genomic DNA was sequenced and analyzed with the ICE tool to quantify indels. Cloning succeeded for all four sgRNAs. Average transfection efficiency was about 4.4% with Lipofectamine and was difficult to reproduce, whereas calcium phosphate yielded reproducible efficiencies above roughly 10%. ICE analysis showed low editing frequencies for sgRNA2–4 (around 5%) and higher editing for sgRNA1 (around 20%). Overall, the findings indicate that calcium phosphate outperforms Lipofectamine in this setup and that sgRNA1 was the most effective guide among those tested.
Glioblastoma multiforme (GBM) er en aggressiv hjernetumor med dårlig prognose. TDGF1 (Cripto-1) regulerer stamcelleadfærd i fosterudviklingen og er ofte opreguleret i kræft, hvor det kan fremme migration og invasivitet. Dette projekt havde til formål at generere et TDGF1‑knockout i GBM‑cellelinjen U87 ved hjælp af CRISPR/Cas9 og samtidig evaluere klonings- og transfektionsmetoder. Fire singleguideRNA (sgRNA) rettet mod exon 3 i TDGF1 blev designet (CrispOR), annealeret og klonet ind i PX461‑plasmider via BbsI‑restriktionskloning. Efter transformation af E. coli blev plasmiderne sekventeret for at bekræfte korrekt indsættelse. U87‑celler blev transfekteret med de klonede plasmider ved brug af enten Lipofectamine eller calciumfosfat, GFP‑positive celler blev FACS‑sorteret, og genomisk DNA blev sekventeret og analyseret med ICE‑værktøjet for at kvantificere indels. Kloningen lykkedes for alle fire sgRNA. Den gennemsnitlige transfektionseffektivitet var omkring 4,4 % med Lipofectamine og var vanskeligt reproducerbar, mens calciumfosfat gav reproducerbare effektivitetstal over ca. 10 %. ICE‑analysen viste lav redigeringsfrekvens for sgRNA2‑4 (omkring 5 %) og højere for sgRNA1 (omkring 20 %). Samlet peger resultaterne på, at calciumfosfat er mere effektivt end Lipofectamine i denne opsætning, og at sgRNA1 var det mest effektive af de testede guides.
[This apstract has been generated with the help of AI directly from the project full text]