1. Molecular cloning

1.1 Transgenic-random insertion

To generate the random insertion transgenic lines, transposase is used to recognize the 3’P-element and 5’P-element and insert the DNA fragment between these two elements randomly into the genome. Any plasmids with 3’P and 5’P can be used as the vectors. White gene driven by hsp70 promoter is always used as the selection marker. If the selection marker needs to be removed later, it can be flanked by two loxP sites and then be removed by Cre cross (Fig. 1A).

In this system, plasmid Δ2-3 can be used to express the transposase.

1.2 Transgenic-site specific insertion

Φ C31 integrase system is usually used to generate the site-specific insertion in Drosophila. In this system, target gene can be cloned into the vector with ΦC31 attB site (Fig. 1B). If the recipient fly has the attP site in the genome, ΦC31 integrase can induce the integration between them and insert the target gene at the locus where the attP site was. Also, white gene can be used as the selection marker.

1.3 Knockout

To generate the knockout fly, CRISPR/Cas9 system is very efficient. The only construct needed is to clone the guide RNA for target gene after the U6 promoter in vector U6b- sgRNA-short (Fig. 1C) [1]. Cas9 mRNA can be transcribed in vitro and injected in fly embryos with sgRNA construct to express the Cas9 protein, or nanos-Cas9 transgenic line can be used as the recipient fly in which Cas9 protein is expressed in the germ line cells [1].

1.4 Knockin

To generate the knockin fly, except the guide RNA construct as in knockout, DNA fragment which is designed to insert into the target locus (“knockin DNA”) should be cloned into the donor DNA vector, flanking with 5’ and 3’ homologous arms (Fig. 1D).

Fig 1. Constructs for different gene manipulation

2. Injection

Collect the embryos from the recipient fly and inject the DNA or RNA mix into the embryos before cellularization.
The injection mix and the recipient fly used for different gene manipulation is listed in Table 1.

3. Candidate screening

Fig. 2 fly crossing and candidate screening

After the injected embryos develop into the adult fly, cross them with w1118 individually. Screen the offspring with selection marker (here for red eyes) (Fig. 2).

Fig. 2 fly crossing and candidate screening [1] Ren X, et al PNAS 2013, 110, 19012-7.