1 Expression of Metabotropic Glutamate Receptors

2 Figure 1. Domain structure of mGluRs investigated here. Scheme of mGluR1 and mGluR6

3 Constructs used in the work Nde I Strep-Tag Hind III 585 Nde I Strep-Tag Hind III 877 His-Tag Figure 2. mGluR6 constructs in pBlunt Figure 3. Map of pCR-Blunt vector

4 Cloning of the H585 and H516 constructs in pMT4 (for COS-1 cells) and pACMV-tetO (for HEK293) 1.The H585 and H516 constructs were cut from pBlunt vector by EcoRI and blunted by Klenow Fragment of DNA Polymerase. 2.The blunted constructs were cloned into blunted pMT4 and pACMV-tetO vectors 3.The clones were checked for the orientation of inserts and sequenced. 4. The following plasmids were obtained: 1) pMT4+H585 2) pMT4+H516 3) pACMV-tetO+H585 4) pACMV-tetO+H516

Figure 4. Western Blot on HIS-tag. Expression checking of H516 construct in COS-1 cells Expression of H516 consruct in COS-1 cells was not observed 1 - His Marker 2 - Cells Lysate 3 - Supernatant, DM 4 - Pellet, DM 5 - Supernatant, OG 6 - Pellet, OG 7 - Supernatant, CHAPS 8 - Pellet, CHAPS 9 - Supernatant, Sarcosyl 10 - Marker II

6 Stable Transfection of HEK293 with H516 and H585 constructs 1.Cells are grown at the following Geneticin concentrations: 300 µg/ml and 1000 µg/ml 2.There are approximately 3 colonies per plate

7 Procedure of mGluR1 repair N-terminusC-terminus Extracellular region Cysteinene-rich region Transmembrane region C-terminus region Mutations: (1) 367 Trp to Arg (2) 511 Lys to Glu (3) 725 Ile to Val 1) PCR 1: Start Primer + Primer Repairing Mutation (1) 2) PCR 2: Product of PCR 1 + Primer Repairing Mutation (2) 3) PCR 3: Product of PCR 2 + Primer Containing EcoNI (~a.a. 629) 4) Cloning of the PCR3 fragment into pBlunt vector 5) Sequencing of the clone (1) 1D4-Tag (2)(3) EcoNI BamHI

8 Procedure of mGluR1 repair (continued) N-terminusC-terminus Extracellular region Cysteinene-rich region Transmembrane region C-terminus region ) Restriction of pBlunt+PCR3 and pBlunt+R522 with EcoNI/MluI 7) Ligation of EcoNI/MluI fragment from pBlunt+R522 with EcoNI/ClaI fragment from pBlunt+PCR3 8) Sequencing of the results (pBlunt+mGluR1strep) (1) 1D4-Tag (2)(3) EcoNI BamHI Strep-Tag + pBlunt+PCR3 EcoNI/MluI fragmentR522 EcoNI/MluI

9 Cloning of mGluR1 in pMT4 and pACMV-tetO vector 1.The mGluR1 construct was cut from pBlunt vector by BamHI and blunted by Klenow Fragment of DNA Polymerase. 2.The blunted fragment was cloned into blunted pMT4 and pACMV-tetO vectors 3.The clones were sequenced.

10 Conclusion 1.H585 and H516 constructs were cloned into pMT4 and pACMV-tetO vectors 2.Expression of H516 construct was checked in COS-1 cells no expression was observed. 3.Stable transfection of HEK293 with H585 and H516 constructs was done. The cells are still growing. 4.Rat mGluR1 construct was repaired.

11 Future work 1.Checking of HEK293 stably transfected with H516 and H Stable transfection of HEK293 with the full-length mGluR1 3.Transient transfection of COS-1 cells with the full-length mGluR1 and H585 construct