2 Material and methods
2.2 Molecular cloning
2.2.7 Vectors used in this study
pDONR201
Figure 7 Map of pDONR201 from INVITROGEN
T2, rrnB T2 transcription termination sequence; T1, rrnB T1 transcription termination sequence; attP1, attachment site; CAM res gene, chloramphenicol resistance gene; attP2, attachment site; Kana res gene, kanamycin resistance gene.
pDONR201 from INVITROGEN was used as a basic cloning vector for cloning with Gateway (section 2.2.3). The Gateway cassette of pDONR201 is flanked by attP sites and contains a chloramphenicol resistance gene and the sequence of the ccdB gene (Bernard and Couturier, 1992; Miki et al., 1992).
In addition, pDONR201 encodes a kanamycin resistance gene (Figure 7).
pCR blunt
pCR Blunt (INVITROGEN) was used together with the Zero Blunt PCR cloning kit from INVITROGEN for sub cloning and used following the manufacturer’s instructions.
L4440
The L4440 vector was created in the laboratory of Andrew Fire, Stanford Departments of Pathology and Genetics, U.S.A., for RNAi in C. elegans (www.addgene.org, Plasmid 1654: L4440). The multiple cloning site of this vector is flanked by two T7 promoters. Transformed in an appropriate E. coli strain, a sequence of choice cloned between these two T7 promoters is transcribed to
double-stranded RNA (section 2.3.8.1).
for RNAi feeding (section 2.3.8.3). For Gateway (www.addgene.org, Plasmid 11344: L4440gtwy)
attR sites (section 2.2.3) was cloned between the T7 promoters using
Figure 8 Map of L4440 from the Fire lab
7, T7 promotor; Amp res gene, ampicillin resistance gene.
Prab-3::GFP::Gateway
This vector is based on the C. elegans
laboratory of Kang Shen, Stanford Institute for Neuro U.S.A., which was modified for this study
comprised an exchange of the promoter sequence against the promoter sequence of was obtained from the plasmid pUH4
Institute, University of Göttingen specific subset of neurons, whereas modified, the newly created vector of C. elegans. The sequence of Prab
Figure 9 Map of Prab-3::GFP::Gateway Amp res gene, ampicillin resistance gene;
In this study, L4440 without an insert was used as negative control .3.8.3). For Gateway cloning, the modified vector
Plasmid 11344: L4440gtwy) was used. In L4440gtwy, a Gateway cassette with .2.3) was cloned between the T7 promoters using EcoRV sites (
Map of L4440 from the Fire lab
ampicillin resistance gene.
C. elegans expression vector pSM Pmig-13::GFP::Gateway from the Stanford Institute for Neuro-Innovation & Translational Neurosciences, which was modified for this study by conventional cloning (section
comprised an exchange of the promoter sequence against the promoter sequence of
pUH4 Prab-3::GFP provided by Stefan Eimer, European Neuroscience
Göttingen. The promoter sequence (P) of mig-13 drives expression only in a whereas the promoter of rab-3 drives pan-neuronal expression. Thus modified, the newly created vector (Figure 9) enables expression of GFP-tagged fusions in all neurons
rab-3::GFP::Gateway can be found in the Appendix.
::GFP::Gateway
mpicillin resistance gene; GW cassette, gateway cassette.
32 In this study, L4440 without an insert was used as negative control cloning, the modified vector L4440gtwy ateway cassette with V sites (Figure 8).
::GFP::Gateway from the Innovation & Translational Neurosciences, section 2.2.4). Modification comprised an exchange of the promoter sequence against the promoter sequence of rab-3, which provided by Stefan Eimer, European Neuroscience drives expression only in a neuronal expression. Thus tagged fusions in all neurons y can be found in the Appendix.
33 Prab-3::mCherry::Gateway
This vector is based on the C. elegans expression vector pSM Pmig-13::mCherry::Gateway (also from the laboratory of Kang Shen), which was modified essentially as described for Prab-3::GFP::Gateway (Figure 9). The sequence of Prab-3::mCherry::Gateway (mCherry, red fluorescent protein) can be found in the Appendix.
pGEX-6P-D21
This vector was created by the laboratory of Erich Wanker at the Max Delbrueck Center for Molecular Medicine, Berlin-Buch, Germany. It is based on the pGEX-6P vector series of AMERSHAM
BIOSCIENCES for IPTG-induced recombinant protein expression in E. coli. pGEX-6P vectors contain a constitutive tac promoter and an ampicillin resistance gene. pGEX-6P-D21 encodes a N-terminal GST-tag in frame with a Gateway cassette (section 2.2.3).
pVV215
pVV215 was used for the expression of HA-tagged proteins in S. cerevisiae and was purchased from the EUROPEAN SACCHAROMYCES CEREVISIAE ARCHIVE FOR FUNCTIONAL ANALYSIS (Frankfurt, Germany). It contains an ampicillin resistance gene and the URA3 gene as a marker for selecting S. cerevisiae transformants. pVV215 encodes the constitutive yeast PGK promoter which is located in front of a Gateway cassette in frame with a C-terminal 3xHA-tag (web.uni-frankfurt.de/fb15/ mikro/euroscarf/
data/pVV215.html).
The following table (Table 3) lists all other vectors used in this study except sub cloning and entry vectors. Sequences cloned for this study were amplified from a C. elegans cDNA library from the laboratory of Stefan Eimer or C. elegans cDNA library clones from OPEN BIOSYSTEM, part of THERMO
FISHER SCIENTIFIC GMBH, Austria.
34 Table 3 Vectors used in this study
vector source description
RNAi feeding
L4440-C34B7.2 this study full length cDNA
L4440-daf-18 OPEN BIOSYSTEMS full length cDNA
L4440-F30A10.6 this study full length cDNA
L4440-F35H12.4 this study full length cDNA
L4440-fem-1 laboratory of Stefan Eimer, ENI,
Göttingen truncated cDNA
L4440-mtm-1 this study full length cDNA
L4440-mtm-3 OPEN BIOSYSTEMS truncated cDNA
L4440-pos-1 Stefan Eimer full length cDNA
L4440-ppk-1 this study full length cDNA
L4440-ppk-2 this study full length cDNA
L4440-ppk-3 OPEN BIOSYSTEMS truncated cDNA
L4440-rab-3 OPEN BIOSYSTEMS truncated cDNA
L4440-snb-1 this study full length cDNA
L4440-unc-26 (5-phosphatase) this study truncated cDNA
L4440-unc-26 (full length) this study full length cDNA
L4440-vps-34 OPEN BIOSYSTEMS full length cDNA
L4440-W09C5.7 this study full length cDNA
L4440-Y75B8A.24 this study full length cDNA
Expression in C. elegans
Prab-3::CB5::CFP Eugenia Butkevich, laboratory of
Dieter Klopfenstein, Göttingen Eimer et al., 2007; CB5 tagged with cyan florescent protein (CFP)
Prab-3::2xFYVE::GFP Eugenia Butkevich FYVE-domain of human early
endosome antigen 1
Prab-3::GFP::PPK-1 this study full length cDNA
Prab-3::aman-2::GFP Eugenia Butkevich full length cDNA, mannosidase II
Prab-3::mCherry::F30A10.6 this study full length cDNA
Prab-3::mCherry::ppk-2 this study full length cDNA
Prab-3::mCherry::ppk-2 (tm3741) this study truncated cDNA Prab-3::mCherry::ppk-2 (ttti8500) this study full length cDNA pFR4 (rol-6 (su1006)) Eugenia Butkevich Cox et al., 1980 Expression in S. cerevisiae
pVV215-sac1 this study full length gDNA
pVV215-F30A10.6 this stuy full length cDNA
Expression in E. coli
pGEX-6P-2 AMERSHAM GST
pGEX-6P-D21-ppk-2 this study wild type PPK-2, N-terminal GST
35