Ward S. Coats

Chapter 8 Use of Recombinant Adenovirus for Metabolic Engineering of Mammalian Cells

Publisher Summary This chapter describes the utility of Adenovirus for transfer of genes involved in metabolic regulation into mammalian cells, with particular emphasis to primary cell types with low replicative activity, such as hepatocytes and cells of the islets of Langerhans. The chapter provides methods and procedures for constructing and propagating new recombinant virions. Recombinant adenoviruses have been useful for delivering genes to whole animals. Viruses have been administered as single injections via accessible blood vessels such as the external jugular vein. Adenovirus in its current form is unlikely to represent the ultimate transfer vector for human gene therapy, given problems such as the lack of integration of the viral genome and the potential for immunological response to injected virus. It is therefore likely that new research initiatives will focus on attempting to engineer “second generation” virions that combine functional features of different viruses—that is, the site-specific integration function of adeno-associated virus (AAV) with the growth and infectivity characteristics of adenovirus.

Replacement of Rbpj With Rbpjl in the PTF1 Complex Controls the Final Maturation of Pancreatic Acinar Cells

BACKGROUND & AIMS The mature pancreatic acinar cell is dedicated to the production of very large amounts of digestive enzymes. The early stages of pancreatic development require the Rbpj form of the trimeric Pancreas Transcription Factor 1 complex (PTF1-J). As acinar development commences, Rbpjl gradually replaces Rbpj; in the mature pancreas, PTF1 contains Rbpjl (PTF1-L). We investigated whether PTF1-L controls the expression of genes that complete the final stage of acinar differentiation. METHODS We analyzed acinar development and transcription in mice with disrupted Rbpjl (Rbpjl(ko/ko) mice). We performed comprehensive analyses of the messenger RNA population and PTF1 target genes in pancreatic acinar cells from these and wild-type mice. RESULTS In Rbpjl(ko/ko) mice, acinar differentiation was incomplete and characterized by decreased expression (as much as 99%) of genes that encode digestive enzymes or proteins of regulated exocytosis and mitochondrial metabolism. Whereas PTF1-L bound regulatory sites of genes in normal adult pancreatic cells, the embryonic form (PTF1-J) persisted in the absence of Rbpjl and replaced PTF1-L; the extent of replacement determined gene expression levels. Loss of PTF1-L reduced expression (>2-fold) of only about 50 genes, 90% of which were direct targets of PTF1-L. The magnitude of the effects on individual digestive enzyme genes correlated with the developmental timing of gene activation. Absence of Rbpjl increased pancreatic expression of liver-restricted messenger RNA. CONCLUSIONS Replacement of Rbpj by Rbpjl in the PTF1 complex drives acinar differentiation by maximizing secretory protein synthesis, stimulating mitochondrial metabolism and cytoplasmic creatine-phosphate energy stores, completing the packaging and secretory apparatus, and maintaining acinar-cell homeostasis.

Analysis of the protein products encoded by variant glucokinase transcripts via expression in bacteria

Five variant transcripts of the single rat glucokinase gene have been described that are naturally expressed in islets of Langerhans, liver and anterior pituitary. Four of these were prepared as cDNA and expressed in bacteria in order to begin to address their physiological roles. Expression of constructs pGKB1 (normal islet/pituitary glucokinase) and pGKL1 (normal liver glucokinase) resulted in glucose‐dependent, glucokinase‐like activity, 7‐fold and 45‐fold, respectively, above background. Expression of pGKB3 (variant islet/pituitary glucokinase) and pGKL2 (variant liver glucokinase) in contrast, did not result in any glucokinase‐like activity.