Identification of differentially expressed genes induced in pancreatic islet neogenesis

Abstract

Cellophane wrapping of the hamster pancreas induces islet neogenesis. We have used the mRNA differential display technique to select for genes expressed during islet neogenesis but not in control pancreata. Ten candidate clones have been identified. Upon sequencing, 6 clones showed a high degree of homology to known genes, 1 showed some, and 3 showed no homology to genes of known sequence. Thus, mRNA differential display is a useful technique to identify genes induced during islet neogenesis, and in combination with screening hamster pancreatic cDNA libraries for full length clones, will enhance the likelihood of capturing the participants in this process.

Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters.

“…Using the mRNA differential display technique, we identified several clones differentially expressed in CW compared with control pancreata (14). One clone, RD19-2, was uniquely expressed in CW as shown by Northern blot analysis (Fig.…”

“…Total RNA was extracted from 10 control and 10 CW hamster pancreata and mRNA differential display technique was performed as previously described (14). Briefly, cDNA was synthesized from 0.2 g of total RNA using 1 M of the four 3 Ј T12VN primers (where V is the degenerate position containing A, C or G, and N is any one of the four deoxynucleotides), 1 ϫ reverse transcription buffer, 25 M each dGTP, dATP, dCTP, dTTP and 100 U of moloney murine leukemia virus reverse transcriptase.…”

“…Total RNA (30 g) was separated on a denaturing gel, transferred to nylon membrane, hybridized, and washed as previously described (14). cDNA probes used in Northern hybridization were labeled through the incorporation of [ ␣ 32 P]dCTP using random primed synthesis to a specific activity of Ͼ 10 9 cpm/ g. A 350 bp RD19-2 cDNA probe was generated by PCR from hamster pancreatic total RNA, a 766 bp INGAP cDNA was cloned in our lab and a 1,000 bp amylase cDNA probe was generously donated by Chris Newgard (University of Texas, Dallas, TX).…”

“…Differential mRNA display technique has been recently described and used to identify differences in subsets of mRNA samples (13). We previously reported on using the mRNA differential display technique and the identification of several clones expressed in regenerating, but not in control pancreata (14). We report here the isolation, sequence, and localization of a novel gene whose protein product is a constituent of Ilotropin, and demonstrate the ability of a unique synthetic peptide corresponding to a region of islet neogenesis associated protein (INGAP) to stimulate proliferation of pancreatic ductal cells in vitro.…”

Prospects and Challenges for Islet Regeneration as a Treatment for Diabetes: A Review of Islet Neogenesis Associated Protein

“…70 Using an mRNA differential display, a novel gene was identified, sequenced, and cloned. 71 The 168-kDa protein product of this gene was found to be a major constituent of the ilotropin extract and to be responsible for the neogenesis activity. 66 This molecule was then named islet neogenesis associated protein.…”