The Paddison Lab uses functional genomics to ask biological questions in mammalian stem and progenitor cells. Our primary goal is to define the biological units of self-renewal, expansion, and lineage commitment in model stem cell systems, including: embryonic stem cells, hematopoietic stem cells, neural progenitor cells, and brain tumor initiating cells. We are particularly interested in understanding the molecular mechanisms that allow stem cells to maintain their unique identity and developmental potential.
What is functional genomics? Functional genomics is the study of the function of genes contained within an organism's genome, or, put another way, an approach to figure out what roles genes have in an organism. [Read more] In the last fifteen years, two powerful homology-based gene targeting technologies have come along that have revolutionized functional genomics in mammals. These are RNAi and CRISPR-Cas9.
RNA interference or RNAi. RNAi emerged out of the pioneering work of Fire, Mello, and colleagues in the nematode Caenorhabditis elegans. Uncovering and characterizing many of the components and biochemical determinants of RNAi in invertebrate systems has helped translate RNAi into a genetic tool in mammals via by RNA-sequence-based targeting of a gene's mRNA. Today, we generally use two types of RNAi triggers to inhibit gene function in mammals: small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs). [Read more] The Paddison Lab routinely performs siRNA and shRNA functional genomic screens in numerous cell types, including human and mouse stem and progenitor cells. As a graduate student, Dr. Paddison helped design some of the first RNAi libraries targeting the human genome.
CRISPR-Cas9. In bacteria, the CRISPR-Cas (Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)–CRISPR-associated (Cas)) pathway acts as an adaptive immune system, conferring resistance to genetic parasites and bacteriophage. The CRISPR-Cas system utilizes a single guide RNA (sgRNA) that is incorporated into a protein effector nuclease (e.g., Cas9) to target exogenous genomic sequences. Unlike RNAi, however, CRISPR-Cas systems are able to target and degrade DNA. This property has been harnessed for sgRNA-directed genome editing in prokaryotes and now eukaryotes. [Read more] The Paddison Lab has performed multiple genome-wide sgRNA screens in human ESCs, hematopoietic cells, neural progenitors, and tumor-derived stem-like cells. We also custom design new CRISPR-Cas9 libraries to probe focused gene sets within the mammalian genome, to identify important protein domains with in genes, and to improve the performance of sgRNA prediction algorithms.
The Paddison Lab, in general, works with four different stem cell systems on a variety of biological questions:
Embryonic stem cells (ESCs) derived from the inner cell mass (ICM) of blastocyst stage mammalian embryos. [Read more]
Human hematopoietic stem and progenitor cells (HSPCs) isolated from peripheral human blood or bone marrow. [Read more]
Human neural progenitor cells (hNPCs) isolated and cultured in conditions resembling their native niches in the adult or developing brain. [Read more]
Glioblastoma multiforme stem-like cells (GSCs) isolated from brain tumor samples in the same defined conditions that permit neural progenitor outgrowth in the lab. [Read more]