Academic journal article Genetics

Successive and Targeted DNA Integrations in the Drosophila Genome by Bxb1 and [Straight phi]C31 Integrases

Academic journal article Genetics

Successive and Targeted DNA Integrations in the Drosophila Genome by Bxb1 and [Straight phi]C31 Integrases

Article excerpt

ABSTRACT At present fC31 is the only phage integrase system available for directionally regulated site-specific DNA integration in the Drosophila genome. Here we report that mycobacteriophage Bxb1 integrase also mediates targeted DNA integration in Drosophila with high specificity and efficiency. By alternately using Bxb1 and fC31, we were able to carry out multiple rounds of successive and targeted DNA integrations in our genomic engineering founder lines for the purpose of generating complex knock-in alleles.

THE serine family of phage integrases such as fC31 are highly useful due to their capability of mediating sitespecific and unidirectional DNA integration in heterologous systems (Groth and Calos 2004). In the past few years, fC31-mediated site-specific DNA integration has gained wide applications in Drosophila for efficient and targeted transgenesis (Groth et al. 2004; Bateman et al. 2006; Bischof et al. 2007; Markstein et al. 2008; Ni et al. 2008). In particular, we and several other groups have developed approaches that combine fC31-mediated DNA integration with gene targeting for achieving directed and efficient modifications of endogenous genomic loci in Drosophila (Gao et al. 2008; Choi et al. 2009; Huang et al. 2009a,b; Weng et al. 2009). For example, in our genomic engineering approach, a "founder line" is first generated by homologous recombination-based gene targeting that effectively replaces the target gene with a fC31-attP ("attPC") integration site. The target locus can then be modified into virtually any desirable knock-in alleles through fC31-mediated integration of corresponding DNA constructs into the founder line (Huang et al. 2009a,b). However, DNA integration effectively destroys the original attPC site by converting it into fC31-attR ("attRC") and fC31-attL ("attLC") sites (Groth and Calos 2004), preventing further DNA integrations into the target locus. Nonetheless, successive DNA integrations into a target locus can be highly desirable when making sophisticated knock-in alleles that are best done by integrating multiple constructs (Ow 2007). Although it is possible to carry out such successive DNA integrations by adding extra attPC or attBC (i.e., fC31- attB) sites on the integration construct, in practice we found that the fC31 often carried out random and promiscuous recombination among multiple attBC and/or attPC sites in Drosophila (J. Huang and Y. Hong, unpublished data), making the process highly inefficient and unreliable. Thus, an additional phage integrase is necessary for successive DNA integrations in a target locus.

Mycobacteriophage Bxb1 integrase (Ghosh et al. 2003; Nkrumah et al. 2006) is a serine integrase that has been shown capable of efficient site-specific integration in heterologous systems, including malaria, plants, and mammalian cells. In addition, characterized Bxb1-attP ("attPX") and Bxb1- attB ("attBX") integration sites not only are distinct from attPC and attBC (Ghosh et al. 2003; Nkrumah et al. 2006), but also are small sizes of 50 bp, which will leave small footprints before and after integrations. To test whether Bxb1 could mediate DNA integration in Drosophila, we made a transgenic vector pAttPX that carries a 52-bp attPX ("attPX-52") (Figure 1A) and a removable w+ marker flanked by loxP sites. Through standard P-element transposition process, we obtained five independent host lines, all coincidently carrying the attPX-52 on the third chromosome. Two of them, the attPX-52#1[w+] and attPX-52#3[w+] lines, were converted to w[2] by excising the w+ marker through Cre/loxP recombination (Figure 1B) (Materials and Methods). We then made a test integration construct, pGE-attBX-GFP, which carries the 46-bp attBX site and a UAS-GFP reporter (Figure 1B), and a construct pET11Bxb1polyA for in vitro synthesis of Bxb1 mRNA (Materials and Methods). pGE-attBX-GFP/Bxb1 mRNA mixtures were prepared and injected into the homozygous attPX-52 embryos using the same protocol of fC31- mediated DNA integration (Groth et al. …

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