Establishment of transgenic cell lines with heterozygous mutations in the PTH1 receptor via CRISPR/Cas9

Introduction Primary failure of eruption (PFE) is a rare, non-syndromic disease which is characterized by an incomplete tooth eruption and associated with mutations in the parathyroid hormone receptor (PTH1R) [1] [2] [3]. However, the cellular background of this disease is not well described. The objective of this study is to establish transgenic cell lines carrying clinical proven mutations to further elucidate the molecular causes and consequences of PFE.

Material and Methods CRISPR/Cas9 was used in a hTERT PDL cell line to establish transgenic cell lines. The sequence of the PTH1R was uploaded into a software which shows all possible guide sequences (gRNA). The gRNA was chosen so that the clinical described mutation was located in the PAM sequence. A HDR template, which is 100 bp long and carries the mutation was designed. The cells were transfected with lipofectamine CRISPRMAX transfection reagent, gRNA, Cas9-Protein and the HDR template for 2 days. Afterwards, single cell colonies were generated to get identical clonal cell lines. Sequencing of the clones was conducted in order to investigate the efficiency of the genome editing.

Results We observed that the CRISPR/Cas9 strategy is working and the transfection efficiency is around 20-40%. If the generated cell lines carry the desired heterozygous mutations at the exact positions is currently validated.

Conclusion In the future we want to generate more transgenic cell lines and use them to investigate functional consequences of the mutations and test the potential of therapeutic strategies.

Publikationsverlauf

Artikel online veröffentlicht:
18. Oktober 2022

• References

• 1 Subramanian H. et al. PLOS ONE. 2016; 11 (11) e0167033
• 2 Decker E. et al. Am J Hum Genet. 2008; 83 (06) 781-786
• 3 Stellzig-Eisenhauer A. et al. J Orofac Orthop. 2010; 71 (01) 6-16