Transcriptional Program of the Endocrine Pancreas in Mice and Humans

Abstract

The Endocrine Pancreas Consortium was formed in late 1999 to derive and sequence cDNA libraries enriched for rare transcripts expressed in the mammalian endocrine pancreas. Over the past 3 years, the Consortium has generated 20 cDNA libraries from mouse and human pancreatic tissues and deposited >150,000 sequences into the public expressed sequence tag databases. A special effort was made to enrich for cDNAs from the endocrine pancreas by constructing libraries from isolated islets. In addition, we constructed a library in which fetal pancreas from Neurogenin 3 null mice, which consists of only exocrine and duct cells, was subtracted from fetal wild-type pancreas to enrich for the transcripts from the endocrine compartment. Sequence analysis showed that these clones cluster into 9,464 assembly groups (approximating unique transcripts) for the mouse and 13,910 for the human sequences. Of these, >4,300 were unique to Consortium libraries. We have assembled a core clone set containing one cDNA for each assembly group for the mouse and have constructed the corresponding microarray, termed "PancChip 4.0," which contains >9,000 nonredundant elements. We show that this PancChip is highly enriched for genes expressed in the endocrine pancreas. The mouse and human clone sets and corresponding arrays will be important resources for diabetes research. Diabetes 52:1604 -1610, 2003 D espite recent progress in ␤-cell biology and diabetes research, tools for the treatment of diabetes have not changed fundamentally. Although it is now clear that islet transplantation is a valuable therapeutic approach, this solution is severely limited by the shortage of islet tissue. Over the past decade, significant advances have been made toward identifying the hierarchy of transcription factors that govern pancreatic development (1). In addition, it has been shown that embryonic stem cells can be differentiated in vitro toward insulin-producing cells, although the issue remains controversial (2-4). Despite these discoveries, major obstacles to the isolation, expansion, and differentiation of pancreatic endocrine stem and/or progenitor cells exist, including a lack of appropriate cell surface antibodies for sorting of progenitor cell populations and an only rudimentary understanding of the lineage of ␤-cells during development and regeneration of the pancreas.To accelerate the progress toward the identification of endocrine precursor cells and factors that regulate the development and differentiation of ␤-cells, the National Institute of Diabetes and Digestive and Kidney Diseases sponsored a program entitled "Functional Genomics of the Developing Endocrine Pancreas" in 1999. The Endocrine Pancreas Consortium was created in response to this program to construct and sequence cDNA libraries derived from multiple stages of pancreatic development. Its purpose was to provide the public expressed sequence tag (EST) databases with sequences from mouse and human endocrine pancreas to discover novel transcripts that could be incorporated into custom m...

Orthogonal analysis of C/EBPβ targets in vivo during liver proliferation

“…For the expression profiling, partial hepatectomies were performed on four wild-type and four C͞EBP␤ Ϫ/Ϫ mutant mice, and liver RNA was extracted at 0, 2, and 16 h after partial hepatectomy. This RNA was reverse-transcribed, fluorescently labeled, and hybridized to the PANCCHIP 5.0 cDNA microarray (22). (Complete results of the PANCCHIP 5.0 and mouse promoter microarray hybridizations are listed in Tables 3 and 4, which are published as supporting information on the PNAS web site.)…”

“…RNA was labeled with amino-allyl dUTP and d(T) 21 to prime reverse transcription. The fluorescent label was coupled to the cDNA and the cDNA was hybridized to PANCCHIP V. 5.0 (22,23). The median intensities of each spot were measured by an Agilent scanner with the GENEPIX software.…”

Reduced Expression of the Insulin Receptor in Mouse Insulinoma (MIN6) Cells Reveals Multiple Roles of Insulin Signaling in Gene Expression, Proliferation, Insulin Content, and Secretion

“…Microarray Sample Pairing-To assess gene expression alteration by comparing two conditions, transcripts from each sample were directly compared using EPCon-pancreatic cDNA microarray with ϳ11,000 probes (27). For each sample pair, transcripts from one condition were labeled with either Cy3 or Cy5 and hybridized with the Cy5-or Cy3-labeled transcripts from the partner condition.…”

Identification of Transcriptional Networks during Liver Regeneration

“…Flourescent label (Cy™Dye, Amersham Biosciences) was coupled to the cDNA and hybridized to the PancChip version 5.0 13K cDNA microarray (23,27). For each array hybridization, labeled liver RNA from one of the four time points was hybridized with a common control RNA sample labeled with a different Cy TM Dye.…”

“…In addition, as a consequence of the complexity and robust nature of the transcriptional changes that occur in response to partial hepatectomy, there remains much to be learned regarding the integration of the products of these genes into the complex signaling and transcriptional hierarchies that result in this highly synchronized regenerative response. In this study, we utilized a cDNA microarray enriched for genes expressed in hepatocytes to profile changes in gene expression in mouse liver cDNA samples isolated 0, 2, 16, and 40 h post-hepatectomy (23,27). We selected these time points to detect differentially expressed genes during the priming phase, mid-G 1 , and peak of S phase in the mouse partial hepatectomy model (see Fig.…”