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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).


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.frontiers-in-genetics.org/flyc31/


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



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 3.1 (2.4)
attP2 8622 y1 w67c23; P{CaryP}attP2 68A4 Red 4.8 (2.1)
attPY 41124 y1 w*/Dp(2;Y)G; P{CaryP}attPY Y Red 7.0 (nd)
*We use the M{vas-int.Dm}ZH-2A as the stable genomic PhiC31 source, except for the X chromosome landing site strains. If you prefer to use the original landings site strains (without the genomic PhiC31 source) please contact us
#FlyBase Drosophila melanogaster (R5.47)
1Eye colors 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.*

BDSC Stock# Genotype Estimated CytoSite# Marker Expression Level Transformation Score6
27387 P{attP.w[+].attP}JB37B 37B7 ? 8.5
27388 P{attP.w[+].attP}JB38F 38F1 ? 5.6
27386 P{attP.w[+].attP}JB53F 53F8 ? 2.6
25091 P{attP.w[+].attP}tara[JB89B] 89B8 ? -
38451 w1118; Dr1/TM3, P{attP.w+.attP}FS10 Sb1 Ser1 79A2 ? 2.0
38452 w1118; Dr1/TM3, Sb1 Ser1 P{attP.w+.attP}FS18 100D1 ? 4.0
38453 y1 w*; snaSco/CyO, P{attP.w+.attP}J04 37B8 ? 4.0
38454 y1 w*; snaSco/CyO, P{attP.w+.attP}J08 37F2 ? 4.0
38455 FM7h, P{attP.w+.attP}FS2/C(1)DX, y1 f1 10B6 ? 4.0
38456 FM7h, P{attP.w+.attP}FS5/C(1)DX, y1 f1 6F3 ? 4.0
*We use the M{vas-int.Dm}ZH-2A as the stable genomic PhiC31 source, except for the X chromosome landing site strains. If you prefer to use the original landings site strains (without the genomic PhiC31 source) please contact us
#FlyBase Drosophila melanogaster (R5.47)
6Transformation efficiency Score was calculated based on actual running orders and will be updated quarterly


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

BDSC Stock# Genotype Estimated CytoSite# Transformant Eye Color1 Transformation Score [Avg. (BACs)]6
97292 PBac{yellow[+]-attP-3B}VK00010 chromosome 2 - - (nd)
9734 PBac{yellow[+]-attP-9A}VK00015 chromosome 2 Red 3.0 (2.4)
9753 PBac{yellow[+]-attP-3B}VK00038 5B8 Red 2.4 (2.4)
9726 PBac{yellow[+]-attP-9A}VK00006 19E7 Light Orange 1.8 (2.4)
9752 PBac{yellow[+]-attP-3B}VK00037 22A3 Orange 4.3 (2.9)
9731 PBac{yellow[+]-attP-9A}VK00012 25A3 Light Orange 0.0 (nd)
9723 PBac{yellow[+]-attP-3B}VK00002 28E7 Red 5.6 (3.1)
9724 PBac{yellow[+]-attP-3B}VK00003ab 31B1, 40A4 Red, Orange 5.0 (3.0)
-12 PBac{yellow[+]-attP-3B}VK00003a 31B1 Red? 0.0 (nd)
-12 PBac{yellow[+]-attP-3B}VK00003b 40A4 Orange? 7.0 (nd)
9730 PBac{yellow[+]-attP-3B}VK00011 40E4 Red 0.3 (0.0)
9733 PBac{yellow[+]-attP-9A}VK00014 43A1 Orange 4.2 (3.6)
9735 PBac{yellow[+]-attP-3B}VK00016 47C6 Orange 2.0 (nd)
9728 PBac{yellow[+]-attP-3B}VK00008 50A3 Light Orange - (nd)
9747 PBac{yellow[+]-attP-9A}VK00030 50E1 Orange 3.1 (2.2)
9736 PBac{yellow[+]-attP-9A}VK00018 53B2 Red 5.1 (1.9)
9740 PBac{yellow[+]-attP-9A}VK00022 57F5 Orange 4.5 (nd)
9722 PBac{yellow[+]-attP-3B}VK00001 59D3 Orange 4.1 (1.5)
97462 PBac{yellow[+]-attP-9A}VK00029 h47-h53 - - (nd)
9748 PBac{yellow[+]-attP-3B}VK00031 62E1 Orange 5.3 (2.2)
9750 PBac{yellow[+]-attP-3B}VK00033 65B2 Red 4.7 (3.7)
9754 PBac{yellow[+]-attP-3B}VK00039 67E4 - 4.8 (nd)
9737 PBac{yellow[+]-attP-9A}VK00019 68D2 Red 4.2 (1.9)
9741 PBac{yellow[+]-attP-9A}VK00023 70A2 Red 4.0 (3.0)
9751 PBac{yellow[+]-attP-3B}VK00036 70C4 Red - (nd)
9725 PBac{yellow[+]-attP-9A}VK00005 75A10 Orange 6.1 (1.8)
9732 PBac{yellow[+]-attP-9A}VK00013 76A2 Red 5.1 (3.3)
9739 PBac{yellow[+]-attP-9A}VK00021 76C5 Orange - (nd)
9727 PBac{yellow[+]-attP-3B}VK00007 82A1 Orange 1.9 (nd)
9749 PBac{yellow[+]-attP-3B}VK00032 85A2 Light Orange 2.0 (0.0)
9755 PBac{yellow[+]-attP-3B}VK00040 87B10 Red 1.2 (nd)
9744 PBac{yellow[+]-attP-9A}VK00027 89E11 Red 5.9 (2.0)
9745 PBac{yellow[+]-attP-9A}VK00028 92F1 Orange 0.8 (2.4)
9743 PBac{yellow[+]-attP-9A}VK00026 96F3 Light Orange 3.2 (nd)
9738 PBac{yellow[+]-attP-9A}VK00020 99F8 Light Orange 5.4 (3.0)
97422 PBac{yellow[+]-attP-9A}VK00024 101F1 - - (nd)
*We use the M{vas-int.Dm}ZH-2A as the stable genomic PhiC31 source, except for the X chromosome landing site strains. If you prefer to use the original landings site strains (without the genomic PhiC31 source) please contact us
#FlyBase Drosophila melanogaster (R5.47)
1Eye colors 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.

BDSC Stock# Genotype Estimated CytoSite# Transformant Eye Color3 Transformation Score [Avg. (BACs)]6
24480 M{3xP3-RFP.attP'}ZH-2A (with M{vas-int.Dm}ZH-102D) 2A3 Orange 2.6 (nd)
24481 M{3xP3-RFP.attP'}ZH-22A (with M{vas-int.Dm}ZH-2A) 22A2 Light Orange 4.9 (3.6)
24482 M{3xP3-RFP.attP'}ZH-51C (with M{vas-int.Dm}ZH-2A) 51C1 Light Orange 6.9 (2.6)
244835 M{3xP3-RFP.attP}ZH-51D (with M{vas-int.Dm}ZH-2A) 51D9 Red 5.9 (1.9)
244844 M{3xP3-RFP.attP}ZH-58A (with M{vas-int.Dm}ZH-2A) 58A3 Light Orange 7.2 (nd)
24485 M{3xP3-RFP.attP'}ZH-68E (with M{vas-int.Dm}ZH-2A) 68E1 Light Orange 7.1 (1.9)
244864 M{3xP3-RFP.attP'}ZH-86Fa (with M{vas-int.Dm}ZH-2A) 86E18 Red 7.0 (2.0)
236487 M{3xP3-RFP.attP}ZH-86Fb (with M{vas-int.B}ZH-102D) 86F8 Red 7.6 (nd)
24749 M{3xP3-RFP.attP}ZH-86Fb (with M{vas-int.Dm}ZH-2A) 86F8 Red 7.1 (2.2)
244874 M{3xP3-RFP.attP'}ZH-96E (with M{vas-int.Dm}ZH-2A) 96E10 Light Orange 2.4 (nd)
236478 M{3xP3-RFP.attP}ZH-102D (with M{vas-int.B}ZH-86Fb) 102D Red 5.0 (nd)
24488 M{3xP3-RFP.attP}ZH-102D (with M{vas-int.Dm}ZH-2A) 102F4 Red 7.1 (nd)
- 24480NF11 ZH-2A 2A3 Orange 2.0 (nd)
- 24481NF11 ZH-22A 22A2 Light Orange - (nd)
- 24482NF11 ZH-51C 51C1 Orange 1.0 (nd)
- 24483NF11 ZH-51D 51D9 Red 6.0 (nd)
- 24484NF11 ZH-58A 58A3 Light Orange 2.0 (nd)
- 24485NF11 ZH-68E 68E1 Light Orange - (nd)
- 24486NF11 ZH-86Fa 86E18 Red 4.0 (nd)
- 24749NF11 ZH-86Fb 86F8 Red 5.0 (nd)
#FlyBase Drosophila melanogaster (R5.47)
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
7Please use the 24749 if no special reason
8Please use the 24488 if no special reason
11M{vas-int.Dm}ZH-2A removed and loxP-3xP3-RFP-loxP 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 2.2 (nd)
attP4 - y1 w67c23 P{CaryP}attP4 12C6 Orange 2.1 (nd)
attP3 32230 y1 w67c23 P{CaryP}attP3 19C4 Light Orange 0.6 (0.9)
attP40 - y1 w67c23; P{CaryP}attP40 25C6 Red 7.2 (4.6)
attP30 - y1 w67c23; P{CaryP}attP30 29C3-29C4 Red - (nd)
attP14 - y1 w67c23; P{CaryP}attP14 36A10 Orange 1.7 (4.4)
attP33 - y1 w67c23; P{CaryP}attP33 50B6 Orange 4.5 (0.9)
attP16 - y1 w67c23; P{CaryP}attP16 53C4 Red 4.2 (2.4)
attP23 - y1 w67c23; P{CaryP}attP23 60C6-60C7 Red - (nd)
attP88 - y1 w67c23; P{CaryP}attP88 64A12 Red - (nd)
attP112 - y1 w67c23; P{CaryP}attP112 68C12-68C13 Red - (nd)
attP10 - y1 w67c23; P{CaryP}attP10 85D7/92B1 Orange 0.7 (nd)
attP64 - y1 w67c23; P{CaryP}attP64 89B9 Red 2.0 (nd)
attP52 - y1 w67c23; P{CaryP}attP52 89B9-89B12 ? - (nd)
attP154 - y1 w67c23; P{CaryP}attP154 97D2 Orange 4.3 (2.4)
*We use the M{vas-int.Dm}ZH-2A as the stable genomic PhiC31 source, except for the X chromosome landing site strains. If you prefer to use the original landings site strains (without the genomic PhiC31 source) please contact us
#FlyBase Drosophila melanogaster (R5.47)
1Eye colors 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 3.5 (nd)
su(Hw)attP6 322329 y1 w* P{nos-phiC31\int.NLS}X; P{CaryIP}su(Hw)attP6 24A2 Red 5.3 (nd)
su(Hw)attP6 347679 y1 w* P{CaryIP}su(Hw)attP6 24A2 Red 5.0 (nd)
su(Hw)attP5 32231 y1 w* P{nos-phiC31\int.NLS}X; P{CaryIP}us(Hw)attP5 50F1 Red 4.6 (nd)
su(Hw)attP5 34765 y1 w* P{CaryIP}su(Hw)attP5 50F1 Red 2.1 (nd)
su(Hw)attP4 34763 y1 w* P{CaryIP}su(Hw)attP4 67E2 Red 1.0 (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 6.0 (nd)
su(Hw)attP1 3556710 y1 w* P{nos-phiC31\int.NLS}X; P{CaryIP}su(Hw)attP1 87B13 Red 4.8 (nd)
&Some lines contain the nos-PhiC31 genomic integrase source as indicated at the genotype description. We use the M{vas-int.Dm}ZH-2A as the stable genomic PhiC31 source if the original site did not contain any PhiC31 genomic source, except for the X chromosome landing site strains. If you prefer to use the original landings site strains (without the genomic PhiC31 source) please contact us
#FlyBase Drosophila melanogaster (R5.47)
1Eye colors 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 5/14/2013

Highlighted are popular/preferred strains


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