Recombinant DNA techniques

The GABABR1α gene fragment was cut out from the pCI-neo vector with Nhe I &

Not I and cloned into the pcDNA5/FRT at the same restriction sites (The new plasmid: pcDNA5/FRT- GABA R1α, see figure 2.5). The GABA R2 gene fragment

was cut with Xho I and inserted to the pBudCE4.1 at the Xho I site (The new plasmid:

npBudCE4.1- GABABR2, see figure 2.5). The new plasmid, pBudCE4.1- GABABR2, was digested with Mlu I and Xba I and ligated to the pcDNA5/FRT-GABABR1α fragment which was cut with Pme I.

The final vector pcDNA5/FRT- GABABR1α-npBudCE4.1- GABABR2 was used to transfect the HEK293 cells. All enzymatic reactions (ligation, dephosphorylation, blunt-end and restriction) were used in order to obtain the final vector are summarized in figure 2.5.

2.2.3.1 GABAB cDNA

The GABAB receptor cDNAs were derived from Novartis Pharma AG. The GABAB

receptor 2 and GABAB receptor 1α from rat were used for the cloning.

2.2.3.2 DNA maxi preparation

For the preparation of larger amounts of high copy plasmid DNA Qiagen Plasmid Maxi Kit (Qiagen, Hilden, Germany) was used. The procedure was carried out according to the manufacturer’s instructions. DNA was stored in 1xTE buffer (10 ml 1 M Tris pH 8.0, 2 ml 0.5 M EDTA pH 8.0, Up to 1 lit with H2O) at 4 °C.

2.2.3.3 DNA sequencing

The sequencing was performed by Fritz Benseler at the Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine in Göttingen, using the Applied Biosystems 373 DNA sequencer according to Sanger et. al. (1977).

Analyses of the sequences were performed using Lasergene software (DNASTAR, Madison, Wisconsin, USA).

2.2.3.4 DNA digestion with restriction enzymes

Restriction analyses were performed as a control for the composition of the DNA (analytical digest) or for cloning purpose (preparative digest).

Analytical digest were performed for 2-3 hours at 37 °C in 15 µl reaction volume.

Analytical digest mixture:

1 µl DNA

1.5 µl 10 x buffer (enzyme specific) 0.5 µl restriction enzyme

H2O up to 15 µl

Preparative digest were performed at 37 °C in 60 µl reaction volume overnight.

Preparative digest mixture:

4 µl DNA

6 µl 10 x buffer (enzyme specific) 2 µl Restriction enzyme (10 U/µl) H2O up to 60 µl

DNA fragments obtained from the restriction digestions were directly analyzed by agarose gel electrophoresis.

2.2.3.5 Purification of DNA

DNA fragments were purified by phenol-chloroform extraction or separated on an agarose gel and the fragments of interest were purified using the QIAquick Gel extraction Kit (Qiagen).

ƒ Purification of DNA from agarose gel (see section 2.2.1.4).

ƒ DNA purification (phenol-chloroform extraction) and DNA precipitation

DNA can be purified by phenol-chloroform extraction to remove proteins from DNA solutions. After restriction digestion or blunting the DNA fragments were purified by phenol-chloroform extraction and then precipitated.

The DNA solution was mixed with 0.1xvolume 3M sodium acetate and 1xvolume phenol-chloroform-isoamyl alcohol (25:24:1) followed by centrifugation at 13.000 rpm for 10 min. The top aqueous phase (supernatant) was collected and 1xvolume of isopropanol was added followed by brief mixing and incubation for 5 min on ice.

After centrifugation for 15 min at 13.000 rpm the supernatant was removed and the pellet was washed with 70 % ethanol. The pellet was dried in air and then re-suspended in 10 µl dH2O.

2.2.3.6 Dephosphorylation of DNA fragments

To prevent the self-ligation of a vector in ligation reactions (e.g. when a plasmid was cut with only one enzyme or blunt-ended), 5´ phosphate groups were removed by hydrolysis. The dephosphorylation was performed using alkaline phosphatase (AP) in 10 x AP dephosphorylation buffer (Boehringer Ingelheim, Germany) for 30 min at 37 °C.

The following reaction mixture was used:

60 µl vector DNA (after preparative digestion) 20 µl 10xAP dephosphorylation buffer

2 µl AP (alkaline phosphatase) 120 µl H2O

2.2.3.7 Blunt-end cloning

Blunt end cloning was performed when no compatible restriction sites were present.

After restriction digestion, dephosphorylation and DNA purification from agarose gel slices, blunting was done using Kleenow Polymerase (Boehringer Mannheim GmbH, Germany).

The linearised vector was dissolved in 21.5 µl dH2O and the following reaction mixture was set up:

21.5 µl DNA

2.5 µl Kleenow Buffer 0.5 µl dNTP 10 mM

0.5 µl Kleenow Polymerase 2 U/µl

The reaction was carried out at 37 °C for 30 min and the DNA fragments were purified by phenol-chloroform extraction and ethanol precipitation. The blunt-ended Vector and fragment were then used for the ligation reaction.

2.2.3.8 Ligation of DNA fragments

DNA ligations were performed by incubating appropriately treated DNA fragments with an appropriately treated vector and T4 DNA ligase in buffer.

The molar ratio of linear vector to insert fragments was ca 1:10. The components in the table below were mixed in a 1.5 ml tube. The mixture was then incubated at 12 °C overnight for ligating reaction. A parallel ligation in the absence of insert DNA was performed as a control.

Composition of the ligating reaction:

1 µl vector DNA 3.5 µl insert DNA 2.5 µl 10x ligase buffer 1 µl T4 ligase (400 U/μl) 17 µl H2O

The ligation mixture was used then for the transformation of E. coli competent cells by electroporation (see section 2.2.3.9).

Figure 2.5. The Cloning of the GABAB R1α and R2 genes. GABAB R1α was cut out from with Nhe I & Not I and cloned into the pcDNA5/FRT. The GABAB R2 gene fragment was cut with Xho I and inserted to the pBudCE4.1. The new plasmids, pBudCE4.1- GABAB R2, was digested with Mlu I and Xba I and ligated to the pcDNA5/FRT-GABAB R1α fragment which was cut with Pme I. The final vector was used to transfect the HEK293 cells.

2.2.3.9 Transformation of competent cells by electroporation

DNA is introduced into competent E. coli cells (Escherichia coli XL1-Blue MRF´) by exposing them to a short-high-voltage electrical discharge. 4µl of plasmid DNA (from ligation mixture diluted 1:4 with dH2O for Mini-prep, or 4 µl plasmid DNA for Maxi-prep) was mixed with 40 µl of electro-competent cells in pre-chilled electroporation cuvette and kept on ice for 1 min. The mixture was pulsed at 2.5 kV in a E. coli pulser (BioRad). The transformed cells were then incubated with 1 ml LB medium at 37 °C for 1 h (shaking at 250 rpm).

For Maxi-prep DNA, 4 ml LB medium with the appropriate antibiotic was added to the cell suspension and incubated for further 4 h. 100 µl of the cells were then added to 500 ml LB medium with the appropriate antibiotic and incubated overnight at 37 °C.

For the mini-Prep DNA, the cell suspension was plated onto LB agar plates containing the appropriate antibiotic and grown overnight at 37 °C.

2.2.3.10 DNA mini preparation

White colonies were isolated from plates using sterile toothpicks and suspended in 5 ml of LB medium with the appropriate antibiotic. The colonies were allowed to grow overnight at 37 °C and 400 rpm. The plasmid DNA was isolated from the cells using the QIAprep Spin Miniprep Kit (Qiagen). Briefly, the mini prep DNA was resuspended in 50 µl of EB buffer (10 mM Tris-HCl pH 8.5) at 4 °C. Purified DNA was analysed by restriction digestion.

2.2.3.11 Estimation of DNA concentration

The concentration of nucleic acids was determined using a spectrophotometer (Eppendorf) by measuring the absorption at 260 nm of 10-fold diluted samples in a quartzcuvette. An OD260=1 in a case of 1 cm cuvette corresponds to 50 μg/ml double-stranded DNA, 33 μg/ml single-double-stranded DNA and 30 μg/ml oligonucleotides (Sambrook et al., 1989).

2.2.4 Culture of HEK293 cells stably transfected with GABABR1α and