The following reagents are for transgene coplacement
(Siegal and Hartl, 1996)
and other applications of Cre recombinase.
Cre recombinase-expressing flies
Four fly stocks are available from the Bloomington stock center:
y1 w67c23; MKRS, P{hsFLP}86E/TM6B, P{Crew}DH2, Tb1
y1 w67c23 P{Crey}1b; nocSco/CyO
y1 w67c23 P{Crey}1b; D*/TM3, Sb1
y1 w67c23; nocSco/CyO, P{Crew}DH1
Cre helper plasmid (pMLS104)
pMLS104, a precursor to the fly transgenic constructs, can be used as a helper plasmid to provide a source of Cre (see, for example, Jasinskiene et al, 2003, Nucleic Acids Research 31:e147 and Oberstein et al, 2005, Nature Methods 2:583). Its construction is detailed in Siegal and Hartl, 1996. Click for the inferred DNA sequence of pMLS104 .
Coplacement vectors
The "waffle" vector, pP[wFl], enables transgene coplacement with white+ as a marker for transformation and Cre- or FLP-mediated excision. Its derivative, pP[SFl], contains a unique SphI site in place of the white+ gene.
Click for a PDF file with a map of pP[wFl] or pP[SFl].
Click for DNA sequences in GCG format of pP[wFl] or pP[SFl].
These sequences are assembled from available sequences of their component parts and have not necessarily been verified. In particular, according to the incomplete sequence of Golic and Lindquist's pP[>whs>] listed on FlyBase, the FRT repeats include SpeI sites. The SpeI site in cloning region 1 of pP[wFl] and pP[SFl] would therefore not be unique, contrary to our original vector maps.
The component parts of pP[wFl] are taken from the following plasmids:
The construction was as follows:
1) 670-bp FRT-containing BamHI-HindIII fragment (blunted with Klenow) from pP[>whs>] cloned into SmaI site of pBSIIKS+ to create pFRT-S; orientation recovered is KpnI-(SmaI)-SspI-XbaI-(SmaI)-SacI
2) FRT-containing BamHI-EcoRI fragment (BamHI blunted with Klenow) from pFRT-S cloned into EcoRV/EcoRI sites of pBSIIKS+ to create pFRT-RV; orientation is SacI-EcoRI-(SmaI)-SspI-XbaI-(SmaI)-(EcoRV)- KpnI
3) pP[^w+^] cut with EcoRI and religated, removing mini-white and 3' loxP site, to create pRIpre
4) pRIpre cut with SalI and religated, removing extra XhoI sites, to create pRIvec
5) EcoRI fragment containing mini-white and 3' loxP site from pP[^w+^] cloned into EcoRI site of pSP72 to create pRIins; orientation recovered is such that KpnI sites flank mini-white
6) pRIins cut with XbaI and religated, removing mini-white, to create pRIins2
7) FRT-containing SacI-EcoRI fragment from pFRT-S cloned into SacI/EcoRI sites of pRIvec to create pRIvecFRT
8) FRT-containing KpnI-EcoRI fragment from pFRT-RV cloned into KpnI/EcoRI sites of pRIins2 to create pRIinsFRT
9) XbaI fragment (blunted with Klenow) containing mini-white from pRIins cloned into PvuII site of pRIinsFRT to create pinsFRT; orientation recovered is such that EcoRI sites flank mini-white and 3' loxP and FRT sites
10) EcoRI fragment containing mini-white, loxP, and FRT from pinsFRT cloned into EcoRI site of pRIvecFRT to create pP[wFl]; correct orientation is as in Figure 7 of Siegal and Hartl (1996)
11) mini-white of pP[wFl] replaced with polylinker from pK19 by digesting pP[wFl] completely with SphI (cuts just 3' of second HindIII site in Figure 7) and partially with EcoRI (to cut only at EcoRI site between first FRT and 5' end of mini-white) and ligating with SphI/EcoRI polylinker fragment from pK19, to create pP[SFl]
Cre recombinase-expressing flies
Four fly stocks are available from the Bloomington stock center:
y1 w67c23; MKRS, P{hsFLP}86E/TM6B, P{Crew}DH2, Tb1
y1 w67c23 P{Crey}1b; nocSco/CyO
y1 w67c23 P{Crey}1b; D*/TM3, Sb1
y1 w67c23; nocSco/CyO, P{Crew}DH1
Cre helper plasmid (pMLS104)
pMLS104, a precursor to the fly transgenic constructs, can be used as a helper plasmid to provide a source of Cre (see, for example, Jasinskiene et al, 2003, Nucleic Acids Research 31:e147 and Oberstein et al, 2005, Nature Methods 2:583). Its construction is detailed in Siegal and Hartl, 1996. Click for the inferred DNA sequence of pMLS104 .
Coplacement vectors
The "waffle" vector, pP[wFl], enables transgene coplacement with white+ as a marker for transformation and Cre- or FLP-mediated excision. Its derivative, pP[SFl], contains a unique SphI site in place of the white+ gene.
Click for a PDF file with a map of pP[wFl] or pP[SFl].
Click for DNA sequences in GCG format of pP[wFl] or pP[SFl].
These sequences are assembled from available sequences of their component parts and have not necessarily been verified. In particular, according to the incomplete sequence of Golic and Lindquist's pP[>whs>] listed on FlyBase, the FRT repeats include SpeI sites. The SpeI site in cloning region 1 of pP[wFl] and pP[SFl] would therefore not be unique, contrary to our original vector maps.
The component parts of pP[wFl] are taken from the following plasmids:
|
pP[>whs>] |
Golic and Lindquist (1989) source of FRT sites in pP[wFl] |
|
pBlueScript II KS+ |
Stratagene used for subcloning in intermediate steps |
|
pSP72 |
Promega used for subcloning in intermediate steps |
|
pP[^w+^] |
derivative of pCaSpeR4 (Pirrotta 1988) mini-white flanked by loxP sites construction detailed in Siegal and Hartl (1996) |
|
pK19 |
Pridmore (1987) plasmid with same polylinker as pUC19,but with Kanamycin resistance instead of Amp |
The construction was as follows:
1) 670-bp FRT-containing BamHI-HindIII fragment (blunted with Klenow) from pP[>whs>] cloned into SmaI site of pBSIIKS+ to create pFRT-S; orientation recovered is KpnI-(SmaI)-SspI-XbaI-(SmaI)-SacI
2) FRT-containing BamHI-EcoRI fragment (BamHI blunted with Klenow) from pFRT-S cloned into EcoRV/EcoRI sites of pBSIIKS+ to create pFRT-RV; orientation is SacI-EcoRI-(SmaI)-SspI-XbaI-(SmaI)-(EcoRV)- KpnI
3) pP[^w+^] cut with EcoRI and religated, removing mini-white and 3' loxP site, to create pRIpre
4) pRIpre cut with SalI and religated, removing extra XhoI sites, to create pRIvec
5) EcoRI fragment containing mini-white and 3' loxP site from pP[^w+^] cloned into EcoRI site of pSP72 to create pRIins; orientation recovered is such that KpnI sites flank mini-white
6) pRIins cut with XbaI and religated, removing mini-white, to create pRIins2
7) FRT-containing SacI-EcoRI fragment from pFRT-S cloned into SacI/EcoRI sites of pRIvec to create pRIvecFRT
8) FRT-containing KpnI-EcoRI fragment from pFRT-RV cloned into KpnI/EcoRI sites of pRIins2 to create pRIinsFRT
9) XbaI fragment (blunted with Klenow) containing mini-white from pRIins cloned into PvuII site of pRIinsFRT to create pinsFRT; orientation recovered is such that EcoRI sites flank mini-white and 3' loxP and FRT sites
10) EcoRI fragment containing mini-white, loxP, and FRT from pinsFRT cloned into EcoRI site of pRIvecFRT to create pP[wFl]; correct orientation is as in Figure 7 of Siegal and Hartl (1996)
11) mini-white of pP[wFl] replaced with polylinker from pK19 by digesting pP[wFl] completely with SphI (cuts just 3' of second HindIII site in Figure 7) and partially with EcoRI (to cut only at EcoRI site between first FRT and 5' end of mini-white) and ligating with SphI/EcoRI polylinker fragment from pK19, to create pP[SFl]