Characterization of an enhancer element for establishing transgenic mouse models for enteric nervous system development

Abstract

Although the bactericidal effects of gastric juice are well established, little is known of the role of components other than hydrochloric acid. Indeed, it has been suggested that gastric acid is the only antibacterial factor in gastric juice. The aim of the study therefore was to compare the effects of human gastric juice, acid and pepsin on bacterial survival. 15 gastric juices obtained from patients undergoing routine gastroscopy or gastric drainage and solutions of HCI at p H values of 1.5-6.0 with or without pig pepsin A were used for 'killing' experiments. E. coli C690 and H.pylori E5 were selected as the test organisms. Suspensions of bacteria (1x106 E coli and1xlO8 H pylori) were pre-incubated with test solutions at 37°C. Samples were taken at 0,5, 10,20 then every 20 mins up to Zhrs, neutralised and the viable colonies counted after culturing at 37"C, 15hrs for E coli and 3 days for H pylori.Growth of bacteria was inhibited at pHs3.5, whereas killing required pH<2.5. 14 of the 15 gastric juices had bactericidal activity at pH3.5. The rate of killing was juice dependent with complete death of E.coli occurring between 5-40mins pre-incubation. In two juices 'killing' was observed at pH6.0. Preincubation with pig pepsin 0.5,l.O and 2.0mg/ml in separate experiments at pH's 2.5, 3.0 and 3.5 showed little bactericidal effect on E.coli C690. However, with H.pylori, the viable colonies were reduced to 50% of the control after 20mins incubation in lmg/ml pepsin at p H 3.5and pH3.0. In conclusion, killing of E.coli can occur in gastric juice at pH's 3.5-6.0 inferring component(s) other than acid and the proteolytic activity of pepsin cause this effect. Whereas, pepsin can facilitate killing of H.pylori.The neural and glial cells of the intrinsic ganglia of the enteric nervous system (ENS) are derived from the hindbrain neural crest at the vagal level. Abnormal development of the vagal neural crest causes aganglionosis of the distal colon, leads to Hirschsprung's disease in humans and megacolon in mice. Several genes have been found to be involved in ENS development. The mouse megacolon mutant Dom was recently found to be due to a mutation in the Sox10 gene. We have in our laboratory a transgenic mouse line carrying the lac2 gene driven by a vagal neural crest-specific enhancer element, b3-IIIa, which was previously identified from the Hoxb-3 gene locus. To assess the tissue-specificity of this element, we examined the expression of lac2 in the transgenic mouse line during the course of ENS development. Our results showed that in the b3-IIIa mouse line, lac2 was expressed in the neural tube at the vagal region at 9.5 dpc. From 10.5 to 14.5 dpc, the IacZ-marked cells appeared as a migrating wave from the vagal region down to the developing gut. The lac2-marked cells first appeared in the foregut at 10.5 dpc, then at the mid-and hindgut by 14.5 dpc. Histological sections of 12.5 to 16.5 dpc gut samples showed that the lac2-marked cells appeared as a concentric ring in the mesenchyme. Using an antibody for neurofilamen...