Generation of GFP fusion proteins via Recombineering (recombination-

In this study a fly line expressing a GFP fusion protein under the control of the endogenous promoter was created using Recombineering (Venken et al., 2008). This method is based on homologous recombination in E.coli using recombination proteins provided from λ phage. Linear DNA with homology in the 5' and 3' ends to a target DNA molecule, which is already present in the bacteria can be introduced into heat-shocked

electrocompetent bacteria. The introduced linear DNA will then undergo homologous recombination with the target molecule.

The bacterial strains used for recombineering (SW102 and SW106) contain a defective λ prophage. The phage genes exo (5’-3’ exonuclease), beta (generate recombination activity) and gam (protects linear DNA from nucleases) are under control of the λPL promoter. This promoter is under tight control of the temperature-sensitive λ repressor cl857. When the bacteria are kept at <32°C, no recombination proteins are produced, but the recombination functions can transiently be supplied by a heat-shock at 42°C. The recombineering strains should always be grown at temperatures below 32°C, or they will loose the prophage.

2.9.6.1 Transformation of BAC clone in SW102 cells

After high copy number induction of the BAC clone harboring the genomic region of the gene of interest and subsequent plasmid preparation (see 2.8.1), 1 µl was transformed into 80 µl electrocompetent SW102 cells (see 2.9.8). The cells were incubated shaking at 30°C for at least 1 h, plated on LB-chloramphenicol plates and grown at 30°C for at least 24 h. Clones harboring the BAC were identified via colony PCR (see 2.9.2).

2.9.6.2 Amplification of GFP selection cassette

The template vector used for the amplification of the GFP selection cassette contains a kanamycin resistance gene, which is flanked by two loxP sites upstream of the GFP open reading frame. The primers for the amplification of the selection cassette each contained a 50 bp long sequence homologous to the destination sequence in the BAC. The forward primer contained the last 50 bp before the stop codon of the gene of interest and the reverse primer contained the first 50 bp of the 3’UTR. For the amplification of the cassette, the following PCR program was used:

Step 1: 98°C 30 sec Step 2: 98°C 10 sec Step 3: 60-72 °C 30 sec

Step 4: 72°C 1 min 30 sec (go back to step 2, repeat 40 times) Step 5: 72°C 10 min

store at 4°C

Every cycle the annealing temperature was increased by 0.3°C and in the last cycle it reached the elongation temperature. The PCR product was purified over an agarose gel and freshly used for recombination.

2.9.6.3 Recombination of GFP selection cassette into BAC containing gene of interest A starter culture of SW102 cells harboring the BAC was grown over night at 30°C. From this culture, two diluted cultures (1:10) were inoculated (induced sample + uninduced control) and grown at 30°C. After the culture reached an OD600 of 0.4 - 0.6, the induced sample was incubated for 15 min at 42°C to activate the recombination functions. The uninduced control sample was kept at 30°C. Next, both samples were shaken in an ice-water slurry for 5 min before the cells were pelleted at 3220 rpm, 4°C for 10 min. The cells were washed twice with 25 ml ice-cold autoclaved ddH2O and each time carefully resuspended by tapping. Then the cells were washed once with 10 % glycerol, spun down for 30 sec at 13.000 rpm and finally resuspended in a final volume of 160 µl 10 % glycerol. These fresh electrocompetent cells were divided into 2 samples and transformed with 100 ng purified GFP selection cassette via electroporation (see 2.9.8).

After shaking at 30°C for 1.5 - 2 h, the cells were plated on chloramphenicol (BAC) + kanamycin (cassette) plates and incubated at 30°C for 36 h.

Recombination events of the selection cassette into the BAC clone were verified via colony PCRs using the primer pairs goi-rec-F + Kan-seq-R and Kan-seq-F + goi-3’-rec-R (Figure 12).

2.9.6.4 Removal of the kanamycin cassette via Cre-recombination

In order to remove the kanamycin cassette from the construct, a second recombination step has to be performed. Only then will the GFP be in-frame with the gene of interest.

For this purpose the SW106 cells, which contain a tightly controlled arabinose-inducible cre gene were used. Cre recombinase can mediate recombination between two identical loxP sites.

After preparing DNA from BAC clones containing the GFP selection cassette, the construct was transformed into electrocompetent SW106 cells and positive clones were again verified by colony PCR. For Cre recombination, a fresh starter culture was inoculated in LB medium supplemented with chloramphenicol, but without kanamycin.

After growing the cells at 30°C until they reached an OD600 of 0.5, 0.1 % of filter-sterilized L-arabinose were added to induce expression of Cre recombinase. The cells were grown for an additional hour and several dilutions were plated on LB plates with chloramphenicol, but without kanamycin. To verify the absence of the kanamycin resistancy gene, 16 colonies were picked and streaked onto LB plates with kanamycin.

This second recombination event was also verified via colony PCR with the primers goi-rec-F and goi-rec-R (Figure 12).

2.9.6.5 Copy number induction of positive clones and large construct preparation

After preparing DNA from clones harboring the BAC with GFP cassette and without the kanamycin cassette, the construct was transformed into electrocompetent TransforMax EPI300 cells (Epicentre). After growing an over night culture at 37°C and diluting it 1:10, CopyControl induction solution (Epicentre) was added (1:1000) and the culture was incubated shaking for 5 h at 37°C. The induction solution induces the expression of a mutant trfA gene in the EPI300 cells. This results in initiation of replication from the oriV high copy origin of the BAC clone and amplification of the construct. If this step is omitted, replication will originate from the low copy oriS. DNA preparation was performed with the QIAGEN Large Construct Kit according to the manufacturer’s instructions. After verification of the construct by sequencing, the construct was send to Genetic Services Inc. (Cambridge, MA) for PhiC31

integrase-Figure 12: Generation of a C-terminal tagged GFP- fusion construct via Recombineering. A selection cassette containing two 50 bp long homology arms, a linker sequence, a kanamycin resistance gene flanked by two loxP sites and the eGFP open reading frame was integrated into the gene of interest (goi) which was located on a BAC clone, by recombination. Afterwards, the kanamycin cassette was removed in a second, cre-mediated recombination step. The primers used for colony PCRs are indicated.