Knockout of Hoxa5 in P19 cells by CRISPR-Cas9: Generation and Characterization

Details

Supervisors

Gofflot, Françoise

Faculty

Faculté des sciences

Degree label

Master [60] en sciences biologiques

Abstract

Hox genes encode HOX transcription factors known primarily for their role in structuring bilateral embryos along their anteroposterior axis and regulating the regional identity of cells and tissues during mammalian embryonic development. Among them, Hoxa5 postnatal expression was characterized in the pre-cerebellar nuclei of the pons and medulla oblongata of mice. A transcriptomic analysis on a postnatal Hoxa5 inactivation mouse model revealed that most of the deregulated genes are involved in synaptic functions of GABAergic and glutamatergic neurons of the brainstem. This suggests a role of HOXA5 in the developing brain in the formation, refinement, and plasticity of pre-cerebellar circuits, presenting it as a candidate regulator of key cerebellar functions such as motor coordination, motor learning, and cognitive functions. The aim of this master’s thesis is to establish a Hoxa5 loss-of-function cell line that will enable the study of HOXA5 synaptic functions in a controlled and reproducible in vitro context. The murine P19 pluripotent cell line has been selected for its ease of manipulation and ability to differentiate into neurons following retinoic acid treatment. Using CRISPR Cas9 gene-editing tool, we targeted Hoxa5 coding sequence and subsequently selected 4 mutant cell lines based on clonality and sequence criteria. To validate Hoxa5 inactivation, we performed RT-qPCR, western blot, and immunofluorescence analyses. RT-qPCR revealed increased Hoxa5 mRNA levels in the mutants. This difference, although not expected, is not surprising and can be explained by hypotheses such as Hoxa5 negative feedback regulation or Hoxa5 long non-coding transcripts deregulation. Western blots and immunofluorescence assay provide insightful information about protein production. An anti-HOXA5 antibody was tested in western blots but was not able to detect the protein. On the other hand, immunofluorescence showed encouraging results as a decrease in signal was highlighted in some mutants as compared to the control, indicating a potential knockout of Hoxa5. While further validation of the candidate cell lines is required, the work presented here provides promising evidence for the establishment of a Hoxa5 loss-of-function P19 cell line, therefore opening the way toward new prospects for the characterization of HOXA5 synaptic functions.