Effect of 5′-Flanking Sequence Deletions on Expression of the Human Insulin Gene in Transgenic Mice

Abstract

Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific transcripts in pancreas, but not in other tissues, 3) the specific immunofluorescence staining of pancreatic islets for human C-peptide, and 4) the synthesis and accumulation of human (pro)insulin in isolated islets. Deletions in the injected DNA fragment of sequences upstream from positions -353, -258, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50% of the total insulin does not alter the normal proportion of mice insulins I and II. These results suggest that expression of the human insulin gene in vivo results from the cooperation of several cis-regulatory elements present in the various deleted fragments. With none of the deletions used, expression of the transgene was observed in cell types other than beta-islet cells.

The potential immunogenicity of human insulin and insulin analogues evaluated in a transgenic mouse model

“…The inbred and Fl-hybrid mice were acclimatized 3 weeks before entering the immunization study at an age of 9 weeks. The transgenic mouse line (line 171) used for breeding in this study has previously been described [14][15][16][17]. Briefly, the transgenic mice are the progeny of the original founder mouse produced by microinj ecting an ll-kilobase human chromosomal DNA fragment including the insulin gene (1430 base pairs) into fertilized mouse eggs [14].…”

“…The transgenic mice have tissue-specific expression in the pancreatic beta cells [16]. Expression of human insulin measured as percent of total insulin in the islets of Langerhans is approximately 50 % [17]. For this study the transgenic mice were backcrossed with normal BALB/c mice.…”

An endocrine-specific element is an integral component of an exocrine-specific pancreatic enhancer.

“…A B-like sequence adjacent to an enhancer core (GTGGAAAA; Weiher et al 19831 is conserved among the human, two mouse, and two rat insulin genes. The B-like and core elements are contained within the 48-bp insulin gene E 1 regulatory domain, which binds one or more islet-specific nuclear factors {Ohlsson and Edlund 1986; Philippe et al 1988) and is necessary for optimal expression in cultured insulinoma cells (Karlsson et al 1987) and in transgenic mice (Fromont-Racine et al 1990). The regulatory region of the glucagon gene contains a potential B element that overlaps the distal part of the 40-bp G1 region, which is critical for specific expression of the glucagon gene (Philippe et al 1988).…”

Comparative Analysis of Insulin Gene Promoters

“…There was also a perception that human insulin promoter constructs would not function in transfected rodent cells. However, these worries proved to be unfounded after it was later shown that there is a very high degree of sequence and functional conservation within the transcription factors that regulate the gene (e.g., 89% identity between rat and human PDX-1) and the human insulin promoter exhibited the expected pattern of activity in transgenic mice (9,10). As a result of the decision to concentrate on a detailed analysis of the rat insulin promoter, most of the literature on the insulin promoter pertains to this promoter.…”