5 Materials and methods
DGRC 2 pJet1.2/blunt Cloning of PCR amplicons Fermentas
5.9 Molecular biological methods
All techniques were performed according to standard protocols (Sambrook et al., 1989), if not mentioned otherwise.
5.9.1 Generation of genomic template DNA for PCR
One fly was sedated, collected into an Eppendorf cup and sacrificed by –20◦C freezing for 15 min. 50 µl of squishing buffer was pipetted into a 0.1 ml glass micro tissue grinder (VWR), the fly was added and homogenized with the corresponding glass pistil, until no more fly structures were detectable. The homogenized fly solution was directly used as template for PCR and stored at –20◦C.
5.9.2 Polymerase chain reaction (PCR)
The polymerase chain reaction (PCR) is used to amplify small amounts of DNA. Following reaction was pipetted:
Template DNA (2 µl homogenized fly, 100-500 ng genomic DNA or 0.5-10 ng plasmid-DNA)
10 µl 5x HF Phusion buffer
1 µl of each oligonucleotide (10 mM) 1 µl dNTPs (10 mM)
0.5 µl Phusion polymerase (NEB)
Water added up to a final volume of 50 µl The PCR was conducted with following steps:
Step Temperature Duration Cycles
Denaturation 98◦C 30 sec 1x
Denaturation 98◦C 10 sec
Primer-annealing 55−72◦C 20 sec 30x
Elongation 72◦C 30 sec/kb (depending on
the length of the target DNA)
Elongation 72◦C 5 min 1x
Storage 4◦C ∞ 1x
All PCRs were conducted in a MyCycler Thermal Cycler (Biorad). Oligonucleotides were ordered from MWG.
5.9.3 Electrophoretic separation of DNA-fragments in agarose gels
DNA-fragments were electrophoretically separated in 1% agarose gels depending on their size. 1 g agarose was added to 100 ml 1x TAE buffer (Tab. 5.4) and brought to boil, until the agarose was fully dissolved. 10 µl Midori Green (Biozym) were added to the warm agarose solution. Midori Green intercalates with DNA enabling visualization in a fluorescent reaction stimulated by UV light. The gel was filled into a gel tray and placed at4◦C for hardening. Samples were provided with loading dye (NEB) and loaded onto the hardened agarose gel, which was submerged in 1x TAE in a gel chamber. Gene ruler ready-to-use (Fermentas) ladder was also loaded on the gel as size reference for DNA fragments. Electrophoresis was conducted at 125 V. An UV Solo TS transilluminator (Biometra) was used for visualization and documentation.
5.9.4 Purification of DNA via gel extraction
DNA fragment were cut from the agarose gel with a clean surgical blade. The NucleoSpin® Extract II kit (Macherey-Nagel) was used for extraction according to manufacturer’s instructions.
5.9.5 Measuring of nucleic acid concentrations
DNA/RNA concentration is determined by the nucleic acid maximum absorption of UV light at 260 nm. An OD260 of 1 corresponds to 47.5 µg dsDNA/ml and 37.5 µg ssDNA/ml.
A NanoDrop ND-1000 spectrophotometer (PEQLAB) was used according to
manufac-turer’s instructions to determine the concentrations of DNA/RNA.
5.9.6 Isolation of plasmid-DNA out of small bacterial cultures (mini prep)
Bacteria of one colony were picked from a LB-agar-plate with a pipette tip and grown in 3 ml LB medium (Tab. 5.6) at 37 ◦C and 180 rpm over night. Plasmid DNA was isolated with the NucleoSpin®Plasmid kit (Macherey-Nagel) according to manufacturer’s instruction.
5.9.7 Isolation of plasmid-DNA out of large bacterial cultures (midi prep)
Bacteria of one colony or a smaller liquid culture were picked and grown in 200 ml LB medium at 37◦C and 180 rpm over night. Plasmid DNA was isolated with the Qiagen Plasmid Plus Midi Kit (Qiagen) according to manufacturer’s instructions.
5.9.8 Restriction digest of DNA
DNA was linearized or isolated using restriction endonucleases. For plasmid DNA lin-earization or DNA fragment isolation 2-3 µg DNA were incubated with 1-2 µl of desired restriction endonuclease, 5 µl 10x restriction buffer, and distilled water in a total volume of 50 µl at37◦C for 15 min according to manufacturer’s instructions.
The digest was arrested by freezing at –20◦C or applying on an agarose gel for elec-trophoresis.
5.9.9 Ligation
DNA fragments were ligated the following using the Quick Ligation Kit (NEB):
10 µl Quick Ligase reaction buffer (2 x) 50 ng vector DNA
37.5 ng insert DNA 1 µl Quick Ligase
Water added up to a final volume of 20 µl
The ligation was incubated at RT for 5 min and chilled on ice before transformation of 50 µl chemically competent cells.
5.9.10 Gibson Assembly
Using the Gibson assembly approach, it is possible to seamlessly assembly multiple DNA fragments with different length (Gibson et al., 2009). Therefore, the NEBuilder® HiFi
DNA Assembly Master Mix was used. Oligonucleotides with appropriate overlaps to the 5’ and 3’ fragments were designed and a PCR was performed as described in section 5.9.2. DNA was purifiedviaan agarose gel as described in section 5.9.4. Linearized vector was prepared and concentration of all fragments including the vector was determinedvia Nanodrop (sec. 5.9.5). Following reaction was pipetted to assemble 2-4 DNA fragments:
100 ng linearized vector
200-300 ng of each DNA fragment 10 µl Gibson assembly master mix 10-X µl dH2O
The reaction was incubated at50◦C for 15 min, when two fragments were assembled and 1 h, when 3-4 fragments were used. 10 µl of the reaction was used to transform chemically
competent cells (sec. 5.9.11).
5.9.11 Transformation of chemically competentE. colicells
50 µl chemically competent TOP10 or DH5αcells (Invitrogen) were thawed on ice for 10 min. 10 µl DNA solution mix were added and the cells were incubated on ice for additional 30 min. Heat shock was performed by transferring the cell containing Eppendorf cups into a42 ◦C water bath for 30 sec. Subsequently, the cell containing Eppendorf cup was transferred back on ice. Cells were shaken at 180 rpm and 37◦C for at least 30 min, after the addition of 300 µl pre-warmed LB medium (Tab. 5.6). The bacterial suspension was plated onto an LB plate containing the according antibiotic for selection.
Bacteria colonies with the internalized recombinant plasmid were obtained on the plate after incubation at37◦C over night.
5.9.12 Generation of plasmid constructs for injection
All generated PCR-products were sequenced (data not shown). Inserts were amplified with primers listed in Table 5.12 and cloned into the desired vectorviaGibson Assembly. The success of a cloning procedure was confirmed by sequencing.
For the Mega project, all double stranded DNA fragments were synthesized by Eurofins Genomics and cloned into thepGE-attBvectorviaeither NsiI-NdeI or XhoI-NsiI restriction sites.
5.9.13 Generation of CRISPR/Cas9 constructs
Clustered regularly interspaced short palindromic repeats (CRISPR) is a genome editting method that allows precise modification of genes. The system is involved in adaptive immunity in bacteria and archea.
A minimal two component system able to induce site-specific cleavage of DNA was identified by (Jinek et al., 2012). FlyCRISPR Optimal Target Finder tool (http://flycrispr.
molbio.wisc.edu/) to identify highly specific target sites in theDrosophilagenome was used to find a target sequence in the gene of interest and design an oligonucleotide pair for Cas9 nuclease cleavage. Cells initiate DNA repair after induced double strand DNA breaks. However, repair mechanisms including non-homologous end joining (NHEJ) are imprecise and often result in insertions and/or deletions at the breaking point with the potential of generating loss-of-function-mutations.
A 20 bp target sequence at the desired breaking point was selected fullfilling following criteria: The target sequence was followed by a 3 nucleotide PAM sequence NGG and begin with a G to optimize U6 driven transcription.
Complementary sense and antisense oligonucleotides were designed, with overhang sequences 5’–CTTC–3’ at the sense oligo and 3’–CAAA–5’ at the antisense oligo. Addi-tionally, both oligos were phosphorylated with T4 polynucleotidkinase.
Following reaction was pipetted for phosphorylation:
1 µl sense oligo (100 µM) 1 µl antisense oligo (100 µM) 1 µl 10x T4 ligation buffer (NEB) 1 µl T4 Polynucleotide Kinase (NEB) 6 µl dH2O
Following thermocycler program was aplied to the reaction:
37◦C for 30 min 95◦C for 5 min
The reacion was cooled down to25◦C at a rate of –0.1◦C/sec.
1 µg pBFv-U6.2vector containing ampicillin resistence was cut using BbsI (NEB) ac-cording to manufacturer’s instructions and purifiedvia the NucleoSpin® Extract II Kit (Tab. 5.2). Following ligation reaction was pipetted:
X µl BbsI digestedpBFv U6.2(50 ng) 1 µl annealed oligo insert
1 µl 10x T4 ligation buffer 1 µl T4 DNA ligase (NEB)
dH2O to 10 µl
The reaction was incubated at 25 ◦C for one hour and was used for transformation of chemically competent 10-beta cells (NEB), according to manufacturer’s instructions.
Inserted gRNA was confirmed by T3 or T7 sequencing. For the generation of plasmids carrying 2 gRNAs, a first gRNA was cloned intopBFv-U6.2vectorviaBbsI restriction sites and a second gRNA was cloned intopBFv-U6.2B, a variant ofpBFv-U6.2carrying a dummy sequence flanked by EcoRI and NotI sites upstream of the U6 promoter . The fragment containing the first gRNA and the U6 promotor was cut from thepBFv-U6.2 vector using EcoRI and NotI restriction sites and cloned intopBFv-U6.2B-gRNA that was previously linearized with EcoRI and NotI (Kondo and Ueda, 2013).
The double gRNA approach was used for homology-directed repair (HDR) when gener-atingattPsite gene substitution. All injections were conducted by BestGene Inc.
5.9.14 DNA sequencing
The sequencing of DNA was performed by Microsynth. For sequencing of PCR-DNA 18 ng/100 base pairs DNA were dissolved in 12 µl water. For sequencing of plasmid DNA 0.5 µg/kb were dissolved in 12 µl water. The desired sequencing primers (3 µl of 10mM primer per sequencing reaction) were directly pipetted into the sequencing solution or were chosen from a standard primer list at Microsynth. All sequencing was conducted at Microsynth.
5.9.15 DNA and protein sequence analysis
DNA and protein sequences were analyzed with DNASTAR 7.0. The National Center for Biotechnology Information (NCBI; www.ncbi.nlm.gov), including the Basic Local Align-ment Search Tool (BLAST), Flybase (www.flybase.org) and UniProt (www.uniprot.org) were used for online database research.
TMHMM2.0 (Transmembrane hidden Markov Model), Center for Biological Sequence Analysis, www.cbs.dtu.dk/services/TMHMM-2.0/, was employed for protein sequence analysis.