Analysis of gene expression in a complex differentiation hierarchy by global amplification of cDNA from single cells

Abstract

Tracking of sibling fates reliably identifies the differentiative potential of a single cell taken for PCR analysis, and demonstrates the existence of a variety of distinct and stable states of differentiative commitment. Global amplification of cDNA from single precursor cells, identified by sibling fates, yields a true representation of lineage- and stage-specific gene expression, as confirmed by hybridization to a broad panel of probes. The results provide the first expression mapping of these genes that distinguishes between progenitors in different commitment states, generate new insights and predictions relevant to mechanism, and introduce a powerful set of tools for unravelling the genetic basis of lineage divergence.

Expression of notch receptors, notch ligands, and fringe genes in hematopoiesis

“…Since Notch signalling is important in cell-fate decisions, it is important to determine the expression of the candidate genes in each ceIl population along the differentiation pathway. This goal was achieved by probing a lineage precursor blot (described in Brady et al, 1995; and in Section 2.7). The lineage precursor blot consists of amplified cDNAs from a hierarchy of hematopoietic precursor cells whose potential is known, from maturing hematopoietic cells, and three fibroblast cell lines.…”

“…The lineage precursor blot is a slot blot of amplified cDNAs made from precursors whose ceil fate is known, fiom maturing cells, and three fibroblast ce11 lines (Brady et al, 1995;described in more detail below). The library was constructed such that it consists of the most 3' untranslated region (3' UTR) of the genes expressed in a particular precursor.…”

“…The lineage precursor blot is a slot blot of amplified cDNAs made fi-om precursors whose ce11 fate is known, maturing ceIl populations, and three fibroblast ceIl lines (slightly modified from Brady et al, 1995; kindly provided by Dr. N. Iscove). The source and culture conditions of the samples on the blots are described in Table 3A and Table 3B.…”

Targets of the Tal1 Transcription Factor in Erythrocytes

“…It was also demonstrated that UBE2H expression is up-regulated during differentiation of erythroblasts to reticulocytes and reduced during terminal differentiation stages (53,54). A similar expression pattern was described for Tal1, which is highly expressed in early erythrocyte stages and down-regulated during later stages of maturation (55,56). These observations may suggest a role of E2-conjugases and specifically UBE2H during late erythrocyte differentiation and a regulation by the hematopoietic transcription factor Tal1.…”

Preferential expression of the vasoactive intestinal peptide (VIP) receptor VPAC1 in human cord blood-derived CD34+CD38− cells: possible role of VIP as a growth-promoting factor for hematopoietic stem/progenitor cells

“…Then, the resultant cDNA solution was mixed with 11 ml of 2 Â tailing buffer containing 0.2 M potassium cacodylate (pH 7.2), 4 mM CoCl 2 , 0.4 mM dithiothreitol (DTT), 3.0 mM dATP, and 10 U of terminal transferase (Boeringer Manheim, Manheim, Germany), incubated at 371C for 15 min, and heated to 651C for 10 min. Aliquots (10 ml) of the above cDNA solutions were subjected to PCR in a 50-ml reaction solution containing a 3 0 -(dT) 24 -containing 60-base primer (KV-(dT) 24 primer: 5 0 -GGTAACTAATAC-GACTCACTATAGGGAGATGAATTC-(dT) 24 -3 0 ) as described by Brady et al 24,25 First-round PCR was performed for 25 cycles (941C for 1 min, 501C for 2 min, 721C for 6 min). After the firstround PCR, 1 ml of the first PCR product was added to 49 ml of the same PCR solution described above, and the second-round PCR was performed for 30 cycles (941C for 1 min, 501C for 1 min, 721C for 2 min).…”