LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMOSTRATION. financed by the program

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1 TÁMOP C-13/1/KONV projekt Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére program keretében finanszírozott LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMOSTRATION financed by the program Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities Dátum / Date: NOVEMBER 29. / NOVEMBER 29, 2017 Helyszín / Place: SZBK, GENETIKAI INT., 604. LAB. / LAB # 604, INST. GENETICS, BRC SZEGED, TEMESVÁRI KRT. 62. Gyakorlati foglalkozás címe / Title of the practical demonstration: INTRODUCTION TO DROSOPHILA GENETICS AND DROSOPHILA TUMOR MODELS Gyakorlatvezető / Demonstrator: Szilárd SZIKORA Biological Research Centre Address: H-6726 Szeged, Temesvári krt. 62. Mail: H-6701 Szeged, POB

2 Module #1: Background Notes / instructions: Thomas Hunt Morgan in the fly lab. In the early 1900s, Thomas Hunt Morgan started to work on fruit flies. By studying Drosophila mutants, Morgan discovered the sex-linked heredity and the principles of genetic recombination, that led him to establish the chromosome theory of inheritance. For his fundamental findings on genetics, he won the Nobel Prize in Physiology or Medicine in As a result of his work, Drosophila became a widely used model organism in genetic research. Why to use flies? A few important reasons: small body size easy to grow low cost short life cycle (about days at room temperature) fully sequenced genome balancer chromosomes versatile and powerful methods for all kinds of gene manipulation males and females are easily distinguished page 2

3 Module #2: Sexual dimorphism A5 A6 John Root 2013 A5 A6 To set up controlled crosses, males and females need to be distinguished: Drosophila melanogaster is a sexually dimorphic species. Males and females can be rapidly recognized based on their morphological differences: -Size: Females usually are about 25% larger than males, but this may vary due to several reasons including age, conditions and genetic background. -Color: In males, the dorsal side of abdominal segments 5-6 (A5-A6) is completely dark, whereas the females exhibit a dark stripe only on the posterior edge of A5-A6 segments (curved arrows). Both genders carry transverse stripes on the dorsal side of each abdominal segment. -External morphology: The posterior end of a female abdomen is pointed whereas the male abdomen is rounded. The most reliable morphological trait is the external genitalia. The male genitalia are larger, darker and more complex than the female (white arrows). Additionally, the forelegs of males carry a patch of bristles, called sexcombs that are not present in females (black arrow). For controlled genetic crosses, females must be separated as virgin females, otherwise they risk to be fertilized by their sibling males. The females are capable of mating 8 hours after emerging from the pupal case, therefore virgins have to be collected before this timepoint. Virgins have a dark spot, called meconium, on the left ventral side of their abdomen which also helps to recognize them. page 3

4 Module #3: Marker mutations Generally used recessive and dominant marker mutations John Root 2013 Marker mutations: After Morgan s discoveries, many visible marker mutations that are alter the morphology of Drosophila organs, for instance wing, bristle, eye etc., have been isolated. The heredity of these mutations can be either dominant or recessive. The dominant mutations are indicated with an uppercase letter (for e.g.: Stubble), while recessive mutations are in lowercase letters (for e.g.: page 4

5 yellow). These marker mutations can be used for the investigation of various biological processes and also in mating scheme design. Notes / instructions- continued: Module #4: Balancer chromosomes Balancer chromosomes, unique to the Drosophila model, are very useful and powerful genetic tools. They can be used to maintain recessive lethal mutations in stable strains. Balancer chromosomes have three important features. They suppress genetic recombination between homologue chromosomes, and they carry at least one dominant marker mutation and at least one recessive lethal or sterile mutation which enables researchers to follow the inheritance of these chromosomes easily. Notes / instructions: page 5

6 Module #5: Targeted gene expression using the UAS/GAL4 system The GAL4/UAS (upstream activating sequence) system is a generally used tool for studying targeted gene expression. It consists of two major components: a UAS regulatory site and the yeast Gal4 transcription activator protein, that is able to activate the transcription of target genes located next to a UAS sequence. The expression of Gal4 can be controlled by cell- or tissuespecific promoters or enhancers that will in turn ensure cell- or tissue- specific expression of the target genes fused to a UAS element. In Drosophila, the two modules can initially be generated as two distinct transgenic lines. In the Gal4 driver line, Gal4 is expressed ectopically but it is not able to activate gene expression in the absence a UAS target gene. Similarly, in the UAS line, in the absence of Gal4 the target gene is inactive. However, if the two lines are crossed, the target gene can be activated in the progeny of the cross that inherits both components. The UAS/Gal4 system Chris B. Helps 1998 The aim of the excercise: Investigation into neuronal and muscle specific gene expression by the UAS/Gal4 system. page 6

7 Module #6: Modeling of tumorigenesis in Drosophila JAK/STAT signaling is evolutionarily conserved in eukaryotes, and it is required for the regulation of various cellular processes. It has been reported that JAK/STAT signaling can be activated by interferons and other extracellular signaling molecules. Upon activation, JAK/STAT signaling is able to induce the transcription of a high number of target genes playing a role in cell growth, differentiation, survival and development. Recently, it has been demonstrated that impaired JAK/STAT signaling can lead to cancer, immune disorders and cardiovascular diseases. The Hop tum mutation, affecting the single Drosophila JAK ortholog, has been reported to cause a leukemia-like phenotype characterized by the abnormal proliferation of blood-cells. This recognition has contributed to the idea that the aberrant activation of JAK-STAT can lead to leukemia in humans. The aim of the excercise: Identification and analysis of tumorigenic blood cells by the Gal4/UAS system. The GFP protein under UAS control (UAS-EGFP strain) will be ectopically expressed by a blood cell specific Gal4 driver (atilla-gal4) in wild type and Hop tum flies. Notes / instructions: page 7