microinjection of drosophila embryos for phic31 integrase mediated embryo transgenesis

phiC31 Transgenesi embrionale mediata dall'integrasi in Drosophila

The fruit fly Drosophila melanogaster is extremely amenable to genetic manipulation and genetic analysis. Transgenic fruit flies are widely used in biological research. Since it was developed in the early 1980s, P-element transposon-mediated transgenesis has been indispensable for Drosophila research1. In some scenarios, other transposons, such as piggyBac and Minos have been used for transgenesis in Drosophila as well2. Transgenes via transposon are randomly inserted into the Drosophila genome, and the expression levels of transgenes at different genomic loci may vary due to position effects and thereby can not be compared2. These drawbacks were overcome by the phiC31-mediated site-specific transgenesis3. The bacteriophage&#160;phiC31 gene encodes an integrase that mediates sequence-specific recombination between the attB and attP sites in Drosophila3. Many attP docking sites have been characterized and are available in the Bloomington Drosophila Stock Center3,4,5,6,7,8,9.
The phiC31 transgenesis system for Drosophila has been widely used by the fly community. Among the attP docking sites, attP18 on the X chromosome, attP40 on the second chromosome, and attP2 on the third chromosome are usually the preferred sites for transgene integration on each chromosome because transgenes at the three sites show high levels of inducible expression while having low levels of basal expression5. Recently, however, van der Graaf and colleagues found that the attP40 site, close to the Msp300 gene locus, and transgenes integrated at attP40 cause larval muscle nuclear clustering in Drosophila10. In another recent study, Duan and colleagues found that the homozygous attP40 chromosome disrupts the normal glomerular organization of the Or47b olfactory receptor neuron class in Drosophila11. These findings suggest that rigorous controls should be included when designing experiments and interpreting data.
Drosophila is an ideal model organism for in vivo interrogation of gene function due to its short life cycle, low cost, and easy maintenance. Genome-wide genetic screening relies on the construction of genome-wide transgenic libraries in Drosophila12,13,14,15. We previously generated 5551 UAS-cDNA/ORF constructs based on the binary GAL4/upstream activating sequence (GAL4/UAS) system, covering 83% of the Drosophila genes conserved in humans in the Drosophila Genomics Resource Center (DGRC) Gold Collection16. The UAS-cDNA/ORF plasmids can be used for the creation of a transgenic UAS-cDNA/ORF library in Drosophila. Efficient embryo microinjection technology can accelerate the creation of transgenic fly libraries.
For embryo microinjection, the needle tip is normally broken against a coverslip17. Thereby, needles frequently become clogged or cause leakage of large amounts of cytoplasm, and when these issues arise, new needles must be employed. Although beveling needles can enhance microinjection efficiency, the grinding solution enters the beveled tip during the grinding process. The grinding solution in the beveled tip does not quickly evaporate naturally; therefore, needles can not be utilized for microinjection directly after beveling. These factors reduce the efficiency of embryo microinjection procedures. Here, using the phiC31-mediated site-specific transgenesis in Drosophila as an example, we present a protocol for embryo microinjection for transgenesis in Drosophila.&#160;To prevent the grinding solution from entering the needle, we utilize an air pump to exert air pressure within the needle, thereby preventing the grinding solution from entering the beveled tip. Beveled needles are ready for sample loading and microinjection directly after beveling.

Autore in primo piano: Ottimizzazione della microiniezione di embrioni per la transgenesi in Drosophila

Microiniezione di embrioni per la transgenesi in Drosophila

Transgenesis in Drosophila is essential for studying gene function at the organism level. Embryo microinjection is a crucial step for construction of transgenic flies. Here, we take the phiC31 integrase-mediated transgenesis in Drosophila as an example and present a detailed protocol for embryo microinjection for transgenesis in Drosophila.Injection needles whose tips are beveled by a grinder do not usually get blocked or elicit large cytoplasmic leakage. However, the grinding solution can enter the beveled tip of a needle during the grinding process and the grinding solution inside the tip does not naturally dry up quickly. Our protocol uses a foot air pump with a pressure gauge to apply air pressure to a needle while grinding its tip.This prevents the grinding solution from siphoning, thereby the grinding solution is prevented from entering the beveled tip, enabling the beveled needle to be used immediately.

Microiniezione di embrioni di Drosophila per la transgenesi embrionale mediata dall'integrasi phiC31

Per iniziare, con un pennello, trasferisci gli embrioni di drosofila declorata su una lunga striscia di agar d'uva. Usa un ago da dissezione per allineare circa 60 embrioni lungo il bordo della striscia con la parte posteriore rivolta verso l'esterno. Ora posiziona un nastro biadesivo lungo 18 millimetri nel senso della lunghezza lungo il bordo di un vetrino coprioggetto.Dopo aver rimosso il supporto, premere delicatamente il vetrino coprioggetto sugli embrioni allineati, trasferire il vetrino coprioggetto con gli embrioni in una scatola contenente un essiccante. Aggiungi olio di carbonio halo su di essi. Per microiniettare gli embrioni, caricare prima due microlitri di DNA in un ago usando una pipetta allungata.Posizionare gli embrioni allineati con olio di carbonio halo al microscopio invertito. Successivamente, utilizzare un micromanipolatore per inserire l'ago nelle estremità posteriori di un embrione. Con il comando a pedale, iniettare con cautela il DNA nell'embrione.Rimuovere l'ago dopo aver completato la microiniezione del primo embrione, quindi spostarlo all'estremità posteriore dell'embrione successivo. Trasferire gli embrioni iniettati in una piastra di agar per l'incubazione. Quando le uova si sono schiuse, prepara una fiala con il cibo.Aggiungere alcune gocce di acqua distillata alla fiala. Con uno stereomicroscopio e un pennello, raccogli le larve schiuse, quindi trasferiscile nella fiala. Incubare la fiala a 25 gradi Celsius con un'umidità compresa tra il 50% e il 60% per 10 giorni.Gli embrioni eccessivamente essiccati appaiono deformati e non possono essere utilizzati per microiniezioni. Gli embrioni poco essiccati perdevano una grande quantità di citoplasma quando venivano punti, riducendo così i tassi di sopravvivenza. Gli embrioni adeguatamente essiccati perdevano solo un piccolo volume, se non nullo, quando venivano iniettati.La linea di drosofila che esprime l'integrasi phiC31 utilizzata per la microiniezione di embrioni aveva gli occhi bianchi. I moscerini anziani di questo genotipo hanno gli occhi rosa derivanti dall'accumulo di proteina fluorescente rossa espressa specificamente nell'occhio. Sono state prodotte con successo mosche transgeniche portatrici di un transgene nel sito di attracco attP2 con occhi arancioni.

microinjection-drosophila-embryos-for-phic31-integrase-mediated

Research

JoVE Journal

Genetics

Microinjection of Drosophila Embryos for phiC31 Integrase Mediated Embryo Transgenesis

Embryo Microinjection for Transgenesis in Drosophila

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope.&#160;Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and&#160;presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection,&#160;a crucial step for creating transgenic flies.

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the ...