Generation of an ABCB5-knockout human melanoma cell line using the CRISPR/Cas9 genome editing-tool

: Study of ABCB5 subcellular localization by cell fractionation

Résumé

ABC transporters are integral membrane proteins mediating the active transport of molecules towards the plasma membrane and playing a role in a wide variety of physiological functions of the cell. Many of these transporters are also implicated in cancer drug resistance. ABCB5 has been described as a marker of skin progenitor cells, melanoma stem cells, and limbal stem cells. It was reported to be a mediator of multidrug resistance in many cancers including melanoma. More recent data also suggest a role of ABCB5 in tumor development and progression. Although eleven transcripts of human ABCB5 have been identified, including two long transcripts named ABCB5FL (full-length) and ABCB5β, the transporter remains little characterized and its subcellular localization has not been validated yet. This research project is divided in two parts. First, it aims to generate an ABCB5-knockout human melanoma cell line to create a powerful tool better characterize the ABCB5 transporter. Second, it aims to use this ABCB5-knockout cell line to pursue the study of the ABCB5 subcellular localization. The CRISPR/Cas9 genome editing tool will be employed to knockout the ABCB5 gene, by removing either a fragment of 53 kb or 125 kb. For that purpose, high quality guide RNAs will be selected to target specific regions of the gene and to limit the risk of off-target effects. The ABCB5-knockout cell lines will be validated at the genomic level by PCR and sequencing, and at the proteic level by Western Blot. The latter will also serve to prove the specificity of the anti-ABCB5 antibodies. The method of cell fractionation described by de Duve et al. will be used to study the ABCB5 subcellular localization.We successfully obtained cell lines with a biallelic 53 kb deletion and cell lines with a monoallelic 125 kb deletion in ABCB5. Both have been validated at the genomic level by PCR and sequencing, and the proteic validation is being performed. We performed a first set of cell fractionation on the MelJuso parental cell line to optimize the method and to establish a first distribution profile of ABCB5. Although we have detected an enrichment of ABCB5 in the nuclear and microsomal fractions, our results remain hypothetical. Indeed, the anti-ABCB5 antibody used in the experiment is a polyclonal antibody, which recognizes many other proteins. After validating the specificity of the antibody by Western Blot, we will perform a second fractionation on both ABCB5-expressing and ABCB5-knockout Mel-Juso cell lines to strenghten the current data on the transporter localization.

la date de réponse	17 janv. 2019
langue originale	Anglais
L'institution diplômante	Universite de Namur
Superviseur	Jean-Pierre Gillet (Promoteur)