| Abstract |
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 ABCB5beta, 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.
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