preparation and collection of drosophila embryos for transgenesis

Drosophila'da phiC31 İntegraz Aracılı Embriyo Transgenezi

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.

Yazar Spotlight: Drosophila'da Transgenez için Embriyo Mikroenjeksiyonunun Optimize Edilmesi

Drosophila'da Transgenez için Embriyo Mikroenjeksiyonu

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.

Transgenez için Drosophila Embriyolarının Hazırlanması ve Toplanması

Başlamak için, bir bezi taşlama solüsyonu ile ıslatın. Bezi iğneli öğütücüye sabitleyin, ardından silikon bir kılcal boru kullanarak bir ayak hava pompasına bir iğne bağlayın. İğneyi öğütücünün iğne tutucusuna monte edin.İğnenin açısını, taşlama plakasına 35 derece bükülecek şekilde ayarlayın. İğne ucunun konumunu, taşlama yüzeyinin hemen üzerine yerleştirilene kadar ayarlamak için mikroskop altındaki kaba kontrol düğmesini kullanın. Ardından, iğnenin iç basıncını inç kare başına 40 pound'da tutmak için ayak hava pompasını kullanın.Ardından, iğne ucunun taşlama plakasına temas etmesini sağlamak için ince kontrol düğmesini kullanın. Üç ila beş saniye öğüttükten sonra, hava kabarcıkları uçtan çıkmaya başladığında iğneyi boşaltın. Ardından, 200 mikrolitrelik bir pipeti bir alkol brülörünün dış alevi ile ısıtın.Pipet ucu erimeye başladığında gerin. Pipet ucunun kapalı ucunu makasla kesin. Drosophila embriyolarını klordan arındırmak için, sinekleri üzüm suyu agar plakalarından bir fırça ile çıkarın.Toplanan embriyoları bir hücre süzgecinde çift damıtılmış su ile durulayın. Hücre süzgecini kağıt mendil ile kurulayın. Daha sonra kurutulmuş süzgeci %30 çamaşır suyu içeren bir Petri kabına koyun.Eşit şekilde karışmasını sağlamak için hücre süzgecini manuel olarak karıştırın. Daha fazla transgenez için bir dizi oluşturmak üzere yüzen embriyoları bir boya fırçası ile seçmeden önce embriyoları iki dakika boyunca çift damıtılmış suda durulayın.

preparation-and-collection-of-drosophila-embryos-for-transgenesis

Research

JoVE Journal

Genetics

Preparation and Collection of Drosophila Embryos for Transgenesis

133 Görüntüleme.  University of South China. Başlamak için, bir bezi taşlama solüsyonu ile ıslatın. Bezi iğneli öğütücüye sabitleyin, ardından silikon bir kılcal boru kullanarak bir ayak hava pompasına bir iğne bağlayın. İğneyi öğütücünün iğne tutucusuna monte edin.İğnenin açısını, taşlama plakasına 35 derece bükülecek şekilde ayarlayın. İğne ucunun konumunu, taşlama yüzeyinin hemen üzerine yerleştirilene kadar ayarlamak için mikroskop altındaki kaba kontrol düğmesini kullanın. Ar...

Video: Transgenez için Drosophila Embriyolarının Hazırlanması ve Toplanması

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 ...