PhiC31 integrase-mediated transgenesis systems

PhiC31 integrase-mediated transgenesis systems are based on the site-specific bacteriophage PhiC31 integrase which mediates sequence-directed, irreversible and highly efficient integration between a bacterial attachment site (attB) and a phage attachment site (attP). Injecting plasmid containing attB site into attP-containing docking site strain(s) with PhiC31 integrase activity makes the resultant stable transformants containing your gene-of-interest between attL and attR sites (irreversible).

We use M{vas-int.Dm}ZH-2A (RFP+, GFP+ phenotype) as the genomic PhiC31 source if the attP site stock does not contain it. If the screening marker is fluorescence-related, we may use the genomic P{nos-phiC31\int.NLS}X source (y+, non-fluorescence), please let us know if this is the case.

For vermilion⁺ markers, e.g. for creating stable attP-gRNA lines, you may actually choose the v⁺ based attP site on autosomal chromosome lines from these below lists. We may setup the crosses to the y^-v^- line and screen for v⁺. You may get the same v⁺ G₂ transformants as using the v^- based attP lines. If v+ transformant  on X chromosome, please choose X chromosomal v- background attP line, such as BDSC#34769. This is just regular Plan H or Plan I with extra vermilion⁺ screening. You may also ask us to keep the resulting gRNA lines and have us cross to the proper Cas9 stock and inject their F1s (additional Plan RG/RH/RI) later.

P{CaryP}
Two attP landing sites were created by the Calos group. These two sites were initially created by the traditional P-element system with yellow marker.

Additional attP landing sites were created by the Perrimon group, and were tested for position effects by luciferase expression on various tissues.

And a strain with P{CaryP}attP at Y chromosome was created by Bellen lab.

See below papers for more info.

PhiC31-RMCE
In June, 2006, the Wu lab published a method that targets constructs to predetermined genomic sites using the PhiC31 integrase system in conjunction with Recombinase Mediated Cassette Exchange (RMCE).

See below paper for more info.

P[acman]
P[acman] system makes use of the combined tools of a conditional amplifiable technique for typical P1 and BAC construct, recombineering, and PhiC31-mediated trangenesis. It has the advantage of delivering large DNA fragments into specific genomic locations with attP site, which were generated by the Bellen lab and markered by yellow. Here we are incorporating the P[acman] system with the work from the Basler group which used genomic PhiC31 integrase source instead of co-injecting the PhiC31 mRNA.

In addition, two genomic BAC libraries ("CHORI-321", 83 kb and "CHORI-322" 21 kb average clone size) have been generated based on P[acman] vector.

Please see below paper and visit the Bellen labCHORI BACPAC Resources for more info:
http://flypush.imgen.bcm.tmc.edu/lab/index.html

https://bacpacresources.org/library.php?id=444

https://bacpacresources.org/library.php?id=445

FlyC31
In FlyC31 system, a library of landing platforms conaining attP site throughout the Drosophila genome were created by the Basler and Karch groups, which are ready for the transgenesis of the construct with attB site. These landing platforms were deliberately designed to be manipulated in vivo by Cre/loxP system after transgenesis to get rid of  of the non-necessary sequences and only keep your DNA fragment flanking with one loxP and one attL site (We do not provide this Cre/loxP service).

In order to use Fluorescence screening marker, Cre/loxP excision were performed to get rid of 3xP3-RFP sequence at these attP site, NF lines.

Please see below paper, the listed stocks and visit the FlyC31 website for more info:

http://www.flyc31.org/

MiMIC
MiMIC is a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes. MiMIC system was developed by the collaboration among the laboratories of Hugo Bellen (Baylor College of Medicine), Roger Hoskins (Lawrence Berkeley National Laboratory) and Allan Spradling (Carnegie Institution of Washington).
Please see below paper and visit the Bellen lab MiMIC page for more info:

http://flypush.imgen.bcm.tmc.edu/pscreen/technique.html

The listed strains do not contain genomic PhiC31 integrase source except indicated

Except indicated, we keep and use the attP stains without PhiC31 source for stable long-term passage. e.g. the Fly31 attP strains do not contian M{vas-int.Dm}ZH-2A. The injected G₀ flies are the F1 of attP stock crossed with PhiC31 source strain.

The PhiC31 system and the Gateway System
One of the "Gateway-PhiC31 system", for example, can be found here. Please note the attP1, attP2, attB1, attB2, etc, at the original Murphys Gateway are of different concepts.

Reference
Groth A, Fish M, Nusse R, Calos MP (2004) Construction of transgenic Drosophila by using the site-specific integrase from phage PhiC31. Genetics 166: 1775

Markstein M, Pitsouli C, Villalta C, Celniker SE, Perrimon N (2008) Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nature Genetics 40: 476
Szabad J, Bellen HJ, Venken KJT (2012) An Assay to Detect In Vivo Y Chromosome Loss in Drosophila Wing Disc Cells. G3 (Bethesda) 2(9): 1095
Bateman JR, Lee AM, Wu C-t (2006) Site-specific transformation of Drosophila via PhiC31 integrase-mediated cassette exchange. Genetics 173: 769
Sun FF, Johnson JE, Zeidler MP, Bateman JR (2012) Simplified Insertion of Transgenes Onto Balancer Chromosomes via Recombinase-Mediated Cassette Exchange. G3 2: 551
Venken KJT, He Y, Hoskins RA, Bellen HJ (2006) PhiC31: a BAC transgenic platform for targeted insertion of large DNA fragments in Drosophila melanogaster. Science 314: 1747
Bischof J, Maeda RK, Hediger M, Karch F, Basler K (2007) An optimized transgenesis system for Drosophila using germ-line-specific PhiC31 integrases. Proc Natl Acad Sci USA 104: 3312
Venken KJT, Schulze KL, Haelterman NA, Pan H, He Y, Evans-Holm M, Carlson JW, Levis RW, Spradling AC, Hoskins RA, Bellen HJ (2011) MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes. Nat Methods 8(9): 737