RESUMEN
Binary expression systems like the LexA-LexAop system provide a powerful experimental tool kit to study gene and tissue function in developmental biology, neurobiology, and physiology. However, the number of well-defined LexA enhancer trap insertions remains limited. In this study, we present the molecular characterization and initial tissue expression analysis of nearly 100 novel StanEx LexA enhancer traps, derived from the StanEx1 index line. This includes 76 insertions into novel, distinct gene loci not previously associated with enhancer traps or targeted LexA constructs. Additionally, our studies revealed evidence for selective transposase-dependent replacement of a previously-undetected KP element on chromosome III within the StanEx1 genetic background during hybrid dysgenesis, suggesting a molecular basis for the over-representation of LexA insertions at the NK7.1 locus in our screen. Production and characterization of novel fly lines were performed by students and teachers in experiment-based genetics classes within a geographically diverse network of public and independent high schools. Thus, unique partnerships between secondary schools and university-based programs have produced and characterized novel genetic and molecular resources in Drosophila for open-source distribution, and provide paradigms for development of science education through experience-based pedagogy.
Asunto(s)
Animales Modificados Genéticamente , Proteínas Bacterianas/genética , Drosophila/genética , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Serina Endopeptidasas/genética , Animales , Secuencia de Bases , Sitios de Unión , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Genes Reporteros , Sitios Genéticos , Recombinación Homóloga , Masculino , Especificidad de Órganos , Posición Específica de Matrices de Puntuación , Unión ProteicaRESUMEN
BACKGROUND: The failing heart exhibits an increased arrhythmia susceptibility that is often attributed to action potential (AP) prolongation due to significant ion channel remodeling. The inwardly rectifying K+ current (IK1) has been reported to be reduced, but its contribution to shaping the AP waveform and cell excitability in the failing heart remains unclear. OBJECTIVE: The purpose of this study was to define the effect of IK1 suppression on the cardiac AP and excitability in the normal and failing hearts. METHODS: We used electrophysiological and pharmacological approaches to investigate IK1 function in a swine tachy-pacing model of heart failure (HF). RESULTS: Terminal repolarization of the AP (TRAP; the time constant of the exponential fit to terminal repolarization) was markedly prolonged in both myocytes and arterially perfused wedges from animals with HF. TRAP was increased by 54.1% in HF myocytes (P < .001) and 26.2% in HF wedges (P = .014). The increase in TRAP was recapitulated by the potent and specific IK1 inhibitor, PA-6 (pentamidine analog 6), indicating that IK1 is the primary determinant of the final phase of repolarization. Moreover, we find that IK1 suppression reduced the ratio of effective refractory period to AP duration at 90% of repolarization, permitting re-excitation before full repolarization, reduction of AP upstroke velocity, and likely promotion of slow conduction. CONCLUSION: Using an objective measure of terminal repolarization, we conclude that IK1 is the major determinant of the terminal repolarization time course. Moreover, suppression of IK1 prolongs repolarization and reduces postrepolarization refractoriness without marked effects on the overall AP duration. Collectively, these findings demonstrate how IK1 suppression may contribute to arrhythmogenesis in the failing heart.