Genome Wide Annotation of SC Determinants
DelES: Chromosome Deletions in ES Cells
In this project we use a newly generated collection of segmental haploid chromosomal deletions which covers, in 1300 different clones, over 25% of the genome of the mouse ES cells. Using a BAC complementation strategy adapted for large scale studies, we have recently identified several regions which are haploinsufficient for ESC differentiation (Fortier et al., PLoS Genetics, 2010). This new resource and method will help elucidate novel determinants of stem cell fate.
Development of hematopoietic stem cell expanding factors
In this program, we screen for proteins and small molecules that have the ability to expand human hematopoietic stem cells. We expose CD34+ human cells to collections of products and determine potential candidates using functional assays in immuno compromized mouse models.
Bilodeau et al. Nat. Methods 2007
Bilodeau et al. PLoS One 2009
Fortier et al., PLoS Genetics 2010
Fortier et al., in revision Nat. Cell Biol. 2014
Genome Wide Annotation of SC Determinants - Selected Publications
1. Genome-Wide Interrogation of Mammalian Stem Cell Fate Determinants by Nested Chromosome Deletions
Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP-based system, we now report the creation and characterization of a collection of ~1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity.
Fortier S, Bilodeau M, MacRae T, Laverdure JP, Azcoitia V, Girard S, Chagraoui J, Ringuette N, Hebert J, Krosl J, Mayotte N and Sauvageau G. (2010). Genome-Wide Interrogation of Mammalian Stem Cell Fate Determinants by Nested Chromosome Deletions. PLoS Genetics 6(12): e1001241.
2. Analysis of Blood Stem Cell Activity and Cystatin Gene Expression in a Mouse Model Presenting a Chromosomal Deletion Encompassing Csta and Stfa2l1
The cystatin protein superfamily is characterized by the presence of conserved sequences that display cysteine protease inhibitory activity (e.g., towards cathepsins). Type 1 and 2 cystatins are encoded by 25 genes of which 23 are grouped in 2 clusters localized on mouse chromosomes 16 and 2. The expression and essential roles of most of these genes in mouse development and hematopoiesis remain poorly characterized. In this study, we describe a set of quantitative real-time PCR assays and a global expression profile of cystatin genes in normal mouse tissues. Benefiting from our collection of DelES embryonic stem cell clones harboring large chromosomal deletions (to be reported elsewhere), we selected a clone in which a 95-kb region of chromosome 16 is missing (Del16qB3Δ/+). In this particular clone, 2 cystatin genes, namely Csta and Stfa2l1 are absent along with 2 other genes (Fam162a, Ccdc58) and associated intergenic regions. Overall, this study presents an optimized approach to globally monitor cystatin gene expression as well as a new mouse model deficient in Stfa2l1/Csta genes, expanding the available tools to dissect cystatin roles under normal and pathological conditions.
Bilodeau M, MacRae T, Gaboury L, Laverdure JP, Hardy MP, Mayotte N, Paradis V, Harton S, Perreault C and Sauvageau G. (2009). Analysis of blood stem cell activity and cystatin gene expression in a mouse model presenting a chromosomal deletion encompassing Csta and Stfa2l1. PLoS One 4, e7500.
3. A retroviral strategy that efficiently creates chromosomal deletions in mammalian cells
Chromosomal deletions, as a genetic tool for functional genomics, remain underexploited for vertebrate stem cells mostly because presently available methods are too labor-intensive. To address this, we developed and validated a set of complementary retroviruses that creates a wide range of nested chromosomal deletions. When applied to mouse embryonic stem cells (ESCs), this retrovirus-based method yielded deletions ranging from 6 kb to 23 Mb (average 2.9 Mb), with an efficiency of 64% for drug-selected clones. Notably, several of the engineered ESC clones, mostly those with large deletions, showed major alteration in cell fate. In comparison to other methods that have also exploited retroviruses for chromosomal engineering, this modified strategy is more efficient and versatile because it bypasses the need for homologous recombination, and thus can be exploited for rapid and extensive functional screens in embryonic and adult stem cells.
Bilodeau M, Girard S, Hebert J, and Sauvageau G. (2007). A retroviral strategy that efficiently creates chromosomal deletions in mammalian cells. Nat. Methods 4, 263-268.