Количественное сравнение СНГ-Регуляторного элемента (CRE) Деятельность в Трансгенные Дрозофилы</em

Summary

Фенотипической изменчивости признаков, может быть результатом мутации в цис-регуляторных элементов (CRE) последовательности, шаблоны управления экспрессии генов. Методы получены для использования в дрозофилы могут количественно сравнить уровни пространственные и временные паттерны экспрессии генов посредничестве изменение или естественные варианты CRE.

Abstract

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism′s program for development¹, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution^2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation^5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics.

Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE′s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster⁷ has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites^8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity^11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols.

Here, we present a general protocol to quantitatively evaluate a CRE′s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE′s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Protocol

Обзор: Это видео демонстрирует протокол способны количественно измерить ген регулирующей деятельности для цис-регуляторных элементов (CRE) последовательностей у дрозофилы (D.) MELANOGASTER. Этот протокол может быть использован для сравнения регулирующей деятельности обла?…

Discussion

цис-регуляторных элементов генома кодирует программу, которая определяет экспрессию генов моделей и тем самым процесс развития ^1, и видных местах для обеих мутаций основной морфологической эволюции ^2-4 и фенотипические вариации для человеческих черт ^5,6,19. Несмотр…