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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 and white marker into attP-containing docking site strain(s) with PhiC31 activity makes the resultant stable w+ transformants containing your gene-of-interest between attL and attR sites (irreversible).

We use M{vas-int.Dm}ZH-2A 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 (non-fluorescence), please let us know if this is the case.

For vermillion+ markers, e.g. for creating stable attP-gRNA lines, you may actually choose any of the vermillion+ based attP 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. This is just regular Plan H or Plan I with extra v+ screening. You may also ask us to keep the resulting gRNA lines and have us cross to any of the Cas9 stocks and inject their F1s (additional Plan R) 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. See below paper for more info.


PhiC31-mediated 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).

Please visit the Wu lab for more info:
http://genepath.med.harvard.edu/WuLab/RMCE


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 Ballen lab and markered by yellow. Here we are incorporating the P[acman] system with the work from the Basler group which used endogenous PhiC31 source instead of co-injecting the PhiC31 mRNA.

Please visit the Bellen lab for more info:
http://flypush.imgen.bcm.tmc.edu/lab/index.html


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 visit the Bellen lab MiMIC page for more info:
http://flypush.imgen.bcm.tmc.edu/pscreen/technique.html


FlyC31
In FlyC31 system, a library of landing platforms containing attP site throughout the Drosophila genome were created by the Basler group, 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 most of the non-necessary sequences and only keep your gene-of-interest flanking with one loxP and one attL site.

Please visit the FlyC31 website for more info:
http://www.flyc31.org/why_phic31.php


Perrimon Strains
Additional attP landing sites were created by the Perrimon group, and were tested for position effects by luciferase expression on various tissues. See below paper for more info.

Please visit the Perrimon lab website for more info:
http://genetics.med.harvard.edu/~perrimon/


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

Bateman JR, Lee AM, Wu C-t (2006) Site-specific transformation of Drosophila via PhiC31 integrase-mediated cassette exchange. Genetics 173:769

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. PNAS 104:3312

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

Knapp J-M, Chung P, Simpson JH (2015) Generating Customized Transgene Landing Sites and Multi-Transgene Arrays in Drosophila Using phiC31 Integrase. Genetics 199: 919



Currently available Calos strains (P{CaryP} strains) at BestGene Inc.

Site name BDSC Stock# Genotype Estimated CytoSite# Transformant Eye Color1 Transformation Score [Avg. (BACs)]6
attP1 8621 y1 w67c23; P{CaryP}attP1 55C4 Red 4.5 (2.2)
attP2 8622 y1 w67c23; P{CaryP}attP2 68A4 Red 5.4 (2.0)
attPY 41124 y1 w*/Dp(2;Y)G, P{CaryP}attPY Y Red 5.9 (nd)
#FlyBase Drosophila melanogaster (R6.10)
1Eye color were determined three days after eclosion (heterozgous state)
6Transformation efficiency Score was calculated based on actual running orders and will be updated quarterly. Average score and >30kb BACs scores were shown


Currently available Wu strains and Bateman strains (PhiC31-RMCE strains) at BestGene Inc.

Site name Reference BDSC Stock# Genotype Estimated CytoSite# Original Stock Eye Color7 Transformation Score6
JB37B 27387 P{attP.w[+].attP}JB37B 37B7 Red 6.0
JB38F 27388 P{attP.w[+].attP}JB38F 38F1 Orange 5.8
JB53F 27386 P{attP.w[+].attP}JB53F 53F8 Orange 2.6
JB89B 25091 P{attP.w[+].attP}tara[JB89B] 89B8 Red 6.3
38451 38451 w1118; Dr1/TM3, P{attP.w+.attP}FS10 Sb1 Ser1 79A2 Orange 2.0
38452 38452 w1118; Dr1/TM3, Sb1 Ser1 P{attP.w+.attP}FS18 100D1 Orange 2.0
38453 38453 y1 w*; snaSco/CyO, P{attP.w+.attP}J04 37B8 Orange 8.0
38454 38454 y1 w*; snaSco/CyO, P{attP.w+.attP}J08 37F2 Red 4.0
38455 38455 FM7h, P{attP.w+.attP}FS2/C(1)DX, y1 f1 10B6 Red 2.3
38456 38456 FM7h, P{attP.w+.attP}FS5/C(1)DX, y1 f1 6F3 Orange 3.0
#FlyBase Drosophila melanogaster (R6.10)
7Eye color were determined three days after eclosion. This is the color of the original stock, not the transformant
6Transformation efficiency Score was calculated based on actual running orders and will be updated quarterly. Average score and >30kb BACs scores were shown


Currently available Bellen strains (P[acman] strains) at BestGene Inc.

Site name BDSC Stock# Genotype Estimated CytoSite# Transformant Eye Color1 Transformation Score [Avg. (BACs)]6
VK10 97292 PBac{yellow[+]-attP-3B}VK00010 chromosome 2 - - (nd)
VK15 9734 PBac{yellow[+]-attP-9A}VK00015 chromosome 2 Red 3.7 (2.4)
VK38 9753 PBac{yellow[+]-attP-3B}VK00038 5B8 Red 2.9 (1.1)
VK06 9726 PBac{yellow[+]-attP-9A}VK00006 19E7 Light Orange 1.8 (1.3)
VK37 9752 PBac{yellow[+]-attP-3B}VK00037 22A3 Orange 4.7 (2.3)
VK12 9731 PBac{yellow[+]-attP-9A}VK00012 25A3 Light Orange 0.0 (nd)
VK02 9723 PBac{yellow[+]-attP-3B}VK00002 28E7 Red 5.9 (3.1)
VK03 9724 PBac{yellow[+]-attP-3B}VK00003ab 31B1, 40A4 Red, Orange 5.0 (3.0)
VK03a -12 PBac{yellow[+]-attP-3B}VK00003a 31B1 Red? - (nd)
VK03b -12 PBac{yellow[+]-attP-3B}VK00003b 40A4 Orange? 8.2 (4.1)
VK11 9730 PBac{yellow[+]-attP-3B}VK00011 40E4 Red 0.3 (0.0)
VK14 9733 PBac{yellow[+]-attP-9A}VK00014 43A1 Orange 5.5 (3.6)
VK16 9735 PBac{yellow[+]-attP-3B}VK00016 47C6 Orange 2.8 (0.0)
VK08 9728 PBac{yellow[+]-attP-3B}VK00008 50A3 Light Orange 5.5 (nd)
VK30 9747 PBac{yellow[+]-attP-9A}VK00030 50E1 Orange 3.3 (1.4)
VK18 9736 PBac{yellow[+]-attP-9A}VK00018 53B2 Orange (maybe variegated) 6.0 (2.7)
VK22 9740 PBac{yellow[+]-attP-9A}VK00022 57F5 Orange 5.0 (3.2)
VK01 9722 PBac{yellow[+]-attP-3B}VK00001 59D3 Light Orange 4.3 (4.3)
VK31 9748 PBac{yellow[+]-attP-3B}VK00031 62E1 Orange 5.9 (2.7)
VK33 9750 PBac{yellow[+]-attP-3B}VK00033 65B2 Red 5.1 (3.8)
VK39 9754 PBac{yellow[+]-attP-3B}VK00039 67E4 - 4.8 (nd)
VK19 9737 PBac{yellow[+]-attP-9A}VK00019 68D2 Red 4.2 (1.9)
VK23 9741 PBac{yellow[+]-attP-9A}VK00023 70A2 Red 4.4 (3.8)
VK36 9751 PBac{yellow[+]-attP-3B}VK00036 70C4 Red - (nd)
VK05 9725 PBac{yellow[+]-attP-9A}VK00005 75A10 Orange 6.2 (2.4)
VK13 9732 PBac{yellow[+]-attP-9A}VK00013 76A2 Red 5.3 (4.4)
VK21 9739 PBac{yellow[+]-attP-9A}VK00021 76C5 Orange - (nd)
VK29 97462 PBac{yellow[+]-attP-9A}VK00029 80 - - (nd)
VK07 9727 PBac{yellow[+]-attP-3B}VK00007 82A1 Orange 2.1 (0.0)
VK32 9749 PBac{yellow[+]-attP-3B}VK00032 85A2 Light Orange 2.0 (0.0)
VK40 9755 PBac{yellow[+]-attP-3B}VK00040 87B10 Red 6.6 (1.2)
VK27 9744 PBac{yellow[+]-attP-9A}VK00027 89E11 Red 6.8 (1.6)
VK28 9745 PBac{yellow[+]-attP-9A}VK00028 92F1 Orange 4.3 (2.0)
VK26 9743 PBac{yellow[+]-attP-9A}VK00026 96F3 Light Orange 3.1 (3.8)
VK20 9738 PBac{yellow[+]-attP-9A}VK00020 99F8 Light Orange 5.2 (4.1)
VK24 97422 PBac{yellow[+]-attP-9A}VK00024 101F1 - 0.0 (nd)
#FlyBase Drosophila melanogaster (R6.10)
1Eye color were determined three days after eclosion (heterozgous state)
2These lines were very hard to transform
6Transformation efficiency Score was calculated based on actual running orders and will be updated quarterly. Average score and >30kb BACs scores were shown
12Donated by Jack Bateman 10/9/2009. Recombined single sites of 9724


Currently available Basler strains (FlyC31 strains) at BestGene Inc.

Site name Reference BDSC Stock# Genotype Estimated CytoSite# Transformant Eye Color3 Transformation Score [Avg. (BACs)]6
ZH-2A 24480 M{3xP3-RFP.attP'}ZH-2A 2A3 Orange 2.8 (0.6)
ZH-22A 24481 M{3xP3-RFP.attP'}ZH-22A 22A2 Light Orange 5.1 (3.4)
ZH-51C 24482 M{3xP3-RFP.attP'}ZH-51C 51C1 Light Orange 7.2 (5.1)
ZH-51D 244835 M{3xP3-RFP.attP}ZH-51D 51D9 Red 6.0 (2.4)
ZH-58A 244844 M{3xP3-RFP.attP}ZH-58A 58A3 Light Orange 7.2 (5.5)
ZH-68E 24485 M{3xP3-RFP.attP'}ZH-68E 68E1 Light Orange 6.8 (3.5)
ZH-86Fa 244864 M{3xP3-RFP.attP'}ZH-86Fa 86E18 Red 7.0 (3.8)
ZH-86Fb 24749 M{3xP3-RFP.attP}ZH-86Fb 86F8 Red 7.1 (4.0)
ZH-96E 244874 M{3xP3-RFP.attP'}ZH-96E (with M{vas-int.Dm}ZH-2A) 96E10 Light Orange 2.5 (nd)
ZH-102D 24488 M{3xP3-RFP.attP}ZH-102D (with M{vas-int.Dm}ZH-2A) 102F4 Red 7.1 (4.0)
24480NF - 24480NF12 ZH-2A 2A3 Orange 2.0 (nd)
24481NF - 24481NF11 ZH-22A 22A2 Light Orange - (0.0)
24482NF - 24482NF11 ZH-51C 51C1 Orange 0.5 (nd)
24483NF - 24483NF11 ZH-51D 51D9 Red 5.4 (nd)
24484NF - 24484NF11 ZH-58A 58A3 Light Orange 1.7 (nd)
24485NF - 24485NF11 ZH-68E 68E1 Light Orange - (0.0)
24486NF - 24486NF11 ZH-86Fa 86E18 Red 3.0 (nd)
24749NF - 24749NF11 ZH-86Fb 86F8 Red 7.7 (nd)
#FlyBase Drosophila melanogaster (R6.10)
3According to FlyC31 website
4These strains are weak; BDSC#24486 is the weakest strain (low survival rate after injection)
5Enhancer trap effect from a nearby gene hibris and is not useful for reporter gene analysis and other applications where ectopic expression is an issue
6Transformation efficiency Score was calculated based on actual running orders and will be updated quarterly. Average score and >30kb BACs scores were shown
11M{vas-int.Dm}ZH-2A removed and loxP-3xP3-RFP-loxP removed version
12M{vas-int.Dm}ZH-102D removed and loxP-3xP3-RFP removed version


Currently available Perrimon strains (P{CaryP} strains) at BestGene Inc.

Site name BDSC Stock# Genotype Estimated CytoSite# Transformant Eye Color1 Transformation Score [Avg. (BACs)]6
attP18 32107 y1 w67c23 P{CaryP}attP18 6C12 Orange 3.4 (0.0)
attP4 - y1 w67c23 P{CaryP}attP4 12C6 Orange 2.1 (0.0)
attP3 32230 y1 w67c23 P{CaryP}attP3 19C4 Light Orange 0.7 (0.7)
attP40 - y1 w67c23; P{CaryP}attP40 25C6 Red 7.2 (4.2)
attP30 - y1 w67c23; P{CaryP}attP30 29C3 Red - (nd)
attP14 - y1 w67c23; P{CaryP}attP14 36A10 Orange 1.7 (1.0)
attP33 - y1 w67c23; P{CaryP}attP33 50B6 Orange 4.8 (1.3)
attP16 - y1 w67c23; P{CaryP}attP16 53C4 Red 4.5 (1.6)
attP23 - y1 w67c23; P{CaryP}attP23 60C7 Red - (nd)
attP88 - y1 w67c23; P{CaryP}attP88 64A12 Red - (nd)
attP112 - y1 w67c23; P{CaryP}attP112 68C12-68C13 Red 4.0 (nd)
attP10 - y1 w67c23; P{CaryP}attP10 85D8 Orange 0.7 (nd)
attP64 - y1 w67c23; P{CaryP}attP64 89B9 Red 4.5 (nd)
attP52 - y1 w67c23; P{CaryP}attP52 89B9-89B12 ? - (nd)
attP154 - y1 w67c23; P{CaryP}attP154 97D2 Orange 5.0 (1.8)
#FlyBase Drosophila melanogaster (R6.10)
1Eye color were determined three days after eclosion (heterozgous state)
6Transformation efficiency Score was calculated based on actual running orders and will be updated quarterly. Average score and >30kb BACs scores were shown


Other strains*

Site name BDSC Stock# Genotype Estimated CytoSite# Transformant Eye Color1 Transformation Score [Avg. (BACs)]6
su(Hw)attP8 32233 y1 w* P{CaryIP}su(Hw)attP8 8E10 Red 3.2 (0.0)
su(Hw)attP6 347679 y1 w* P{CaryIP}su(Hw)attP6 24A2 Red 3.0 (nd)
su(Hw)attP5 34765 y1 w* P{CaryIP}su(Hw)attP5 50F1 Red 3.2 (nd)
su(Hw)attP4 34763 y1 w* P{CaryIP}su(Hw)attP4 67E2 Red 2.0 (nd)
su(Hw)attP2 347619 y1 w* P{CaryIP}su(Hw)attP2 92D9 Red 3.5 (nd)
su(Hw)attP1 3476010 y1 w* P{CaryIP}su(Hw)attP1 87B13 Red 5.6 (nd)
VIE260B VDRC#60100 P{attP,y+,w3'}VIE-260B 25C6 Red 8.7 (nd)
JK22C aka JMK-96 - w1118; P{CaryP}JK22C 22C3 Red 2.6 (0.0)
JK73A aka JMK-97 - w1118; P{CaryP}JK73A 65C3 Red 4.4 (0.0)
JK65C aka JMK-98 - w1118; P{CaryP}JK65C 66B4 Red 4.6 (0.0)
JK66B aka JMK-99 - w1118; P{CaryP}JK66B 73A9 Red 6.4 (1.2)
*Some lines contain the nos-PhiC31 genomic integrase source as indicated at the Genotype.
#FlyBase Drosophila melanogaster (R6.10)
1Eye color were determined three days after eclosion (heterozgous state)
6Transformation efficiency Score was calculated based on actual running orders and will be updated quarterly. Average score and >30kb BACs scores were shown
9This strain has very high non-specific integration rate. Service W (PCR confirmation of integration site) is highly recommended
10Service W is NOT available for this site

 

updated 4/17/2017

Highlighted are popular/preferred strains


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