Key words :
intracellular signaling pathways
Functional Genomic Screens in Stem Cells Using Clonal Microarrays
10 Oct, 2007 12:18 pm
The field of functional genomics explores the various functions of genetic sequences within the human genome. Gene function discoveries provide researchers with greater understanding of the instructions a cell receives from its genetic code, and could ultimately lead to advancements in medicinal therapies for numerous diseases.
Due to the large amount of information contained in genomes, high throughput methods to efficiently screen the function of numerous genetic sequences can greatly aid scientist. To discover genes that induce or antagonize a particular cell behavior, researchers can make a library of genetic sequences (102‐106 different sequences), insert the library sequences into the genome of cells, and observe the resulting effect of the inserted genetic sequence on a cell’s behavior. Observations of cell behavior can be performed on a cell by cell basis, and to facilitate automated microscopic analysis of cell behavior, several researchers have recently developed cell‐based microarrays – patterns of cell populations (1 or more cells per pattern feature) spatially distributed on the micron scale on a standard microscope slide. Using cell‐based microarrays, these researchers have screened the effect of thousands of genetic sequences on cell behavior within the space of a single microscope slide (7.5 x 2.5 cm).
Such cell‐based microarrays have been constructed using costly microarrayer machines which spot islands of genetic material or viral particles containing genetic material on a microscope slide such that each island contains a different genetic sequence from the library. Viral particles containing genetic sequences are used to facilitate insertion of the genetic sequence into stem cells, which do not readily uptake exterior genetic material. Cells are then seeded onto the printed microscope slides, and upon adhering on top of an island presenting genetic material or viral particles containing genetic material, they incorporate the underlying genetic sequences into their own genome. After an incubation period, the behavior of each cell population on the microarray can be screened, and the genetic sequences that induced the desired cell behaviors can be identified by the cell population’s position on the microarray. As a prerequisite to constructing this type of cell‐based microarray, highly concentrated genetic solutions must be produced for each genetic sequence in the library. This is likely to be time consuming for genetic libraries of appreciable size. To address this limitation, we have developed a “clonal microarray” method.
We have demonstrated that this clonal microarray method can be used to screen a large genetic library in a high throughput fashion in both adult and embryonic stem cells. In our method, micropatterned surfaces are constructed by using inexpensive, quick, procedurally facile soft‐lithographic methods to microprint regions of a defined surface chemistry. The resulting patterned surface contains an array of cell‐adhesive islands separated by cell‐repellent regions. Prior to seeding the stem cells on the patterned microscope slide, they are infected with viral particles containing genetic sequences from the library in a manner such that only one genetic sequence is inserted into the genome of each infected cell. The stem cells are then seeded onto the patterned surfaces at a density such that 0 or 1 cell adheres to a cell‐adhesive island. Due to the ability of stem cells to self‐renew or proliferate to produce identical daughter cells, the spatially isolated stem cells can either be screened as a single cell, or they can be allowed to self‐renew for several days to produce clonal populations – where all cells in the population are genetically identical because they are descendents of a single cell. After screening, select stem cell populations can be isolated from the microarray, genetically probed to identify the inserted genetic sequence responsible for the stem cells’ behavior, and further propagated to produce a clonal stem cell line with the desired cell behavior. Clonal microarrays can be used to screen over 3500 genetic sequences on a standard microscope slide. In our study, we have used the clonal microarray method to identify genetic sequences that can enhance the proliferative capabilities of stem cells. The clonal microarray method could be widely applied to explore functional genomics in many different cell types.
Ashton RS, Peltier J, Fasano CA, O'Neill A, Leonard J, Temple S, Schaffer DV, Kane R. High-Throughput Screening of Gene Function in Stem Cells using Clonal Microarrays
Stem Cells. Stem Cells , first published online, August 2, 2007.
Abstract available at: http://stemcells.alphamedpress.org/cgi/content/abstract/2007-0468v1