Development of a MACS-based strategy for isolating rare cell populations from animal tissue for transcription factor studies
Two major challenges in studying specific cell populations of embryos or adult organs are: a) getting the sample sufficiently pure for the cells of interest; b) getting sufficient quantities of these cells. Current methods used for isolating specific cell populations from tissues are either too costly or technically challenging to obtain large quantities of samples, or do not yield sufficiently pure samples. We addressed these challenges by developing a Magnetic Activated Cell Sorting(MACS)-based strategy to isolate rare cells of interest from complex animal tissue. We designed a two-component transgenic cell surface protein, BAP-Lngfr, so that two rounds of MACS can be performed --- the first round against the biotinylated BAP (Biotin Acceptor Peptide) component, and the second against the Lngfr component. Having a second round of sorting doubles the purity of the isolated cell sample, so that rare cell populations can be isolated to sufficient purity for sensitive downstream assays. We termed this strategy the `Two-step MACS?, and showed that a rare population of cells (~1% of the 13.5 d.p.c mouse embryo) could be enriched to a sufficiently high purity (~85%) in this way. Sufficient quantities of cell sample can be achieved because the low cost, speed and technical ease of the Two-step MACS makes it scalable. A third challenge emerges when the cells of interest do not express a known cell surface marker. In these circumstances standard cell sorting approaches cannot be used. The method we have developed in this thesis allows cells of interest to be defined by their expression of one or two specific genes of interest rather than known cell surface markers. We achieved this by ensuring the expression of BAP-Lngfr mirrored that of our gene of interest, Sox9. We placed the expression of BAP-Lngfr under the promoter control of Sox9 by gene targeting the Sox9 locus in ES cells, from which transgenic animal tissue can be derived. Sox9 was chosen as our modeltranscription factor of interest for its biological importance as the master regulator of chondrogenesis and for its known expression pattern. Our results demonstrate that even when only one-round of MACS was performed, the resulting enriched sample was sufficient for microarray transcription profiling to elucidate gene regulatory networks in Sox9-expressing cells.
sorting, isolation, enrichment, microarray, single-celled, sox9
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