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Table of contents
Introduction
I. Early development & embryonic stem cells
A. Early mouse embryo development
B. The establishment of pluripotency in vitro
C. Origin and properties
D. The spectrum of pluripotent cells
E. The maintenance of the pluripotent state
II. Signalling pathways regulating pluripotency
A. LIF signalling
B. FGF signalling
C. Wnt signalling
III. Transcription Factors-based regulation of pluripotency
A. Pluripotency factors
B. Oct4 (Pou5f1)
C. Sox2
D. Nanog
E. LIF independent self-renewal
F. Otx2
IV. Polycomb regulation and bivalent domains in pluripotency
A. Polycomb complexes and functions
B. Bivalent domains and Polycomb regulation in pluripotent cells
V. Long non-coding RNAs & mouse ES cells
A. Description and characteristics
B. LncRNAs function in mouse ES cells
VI. From CRISPR discovery to CRISPR activators
A. Historical background
B. Summarized way of action
C. Genome engineering
D. Other kind of versatile DNA-binding protein
E. Transcriptional modulation
F. Examples of CRISPRa studies in stem cell biology and cell reprogramming
A. sgRNA cloning
B. Bio-informatics analysis with Seqmonk program (LASER selection)
C. Cell fractionation
D. Single cell sorting
E. Generation of LASER 23 KO ES cells
VII. Adaptation of the CRISPRa SunTag system in mouse ES cells
A. Why developing CRISPR-activation?
B. Construction of the first SunTag cell line generation
C. Construction of the second SunTag ES cell line generation
D. D. Transcriptional Induction tests
E. Discussion
VIII. The molecular logic of Nanog-induced self-renewal
IX. LASER: LncRNAs Associated with SElf-Renewal of mouse ES cells
A. Introduction
B. Preliminary selection of lncRNAs candidates
C. Characterization of the 24 LASER
D. LASER gRNAs design and test
E. LASER overexpression upon LIF withdrawal
F. Transcriptomic response upon three LASER overexpression
G. Additional lncRNAs candidates selection
H. LASER 23 (Gm14820) characterization
I. Discussion
X. A serendipity-driven approach
A. Introduction
B. An unexpected 2 cell-like state induction
C. First hypothesis: a LASER 1-mediated effect
D. Second hypothesis: an off-target effect
E. Identification of candidate genes
F. Discussion
References
