Expression of Metabotropic Glutamate Receptors
Alexander Yaroshchuk
1. Introduction
2. Methods
3. Results
4. Discussion
1. Introduction
The aim of this work was to develop an expression system for specific constructs
of the Human Metabotropic Glutamate Receptor 6 (HmGluR6) and Rat
Metabotropic Glutamate Receptor 1a (RmGluR1a). On the Fig. 1. the domain
structures of the receptors are shown.
Figure 1. Domain structure of mGluRs.
On the Fig. 2. the constructs used in the work are shown
Figure 2. Constructs used in the work.
2. Methods.
1) Transient transfection of COS-1.
COS-1 confluent 15 cm dishes were used for the experiment.
1. The plates were washed with 25ml of DMEM+Pen/Strep.
2. To each plate 15ml of the following buffer was added: 0.25mg/ml DEAEDextran, 0.1 M Tris-HCl pH 8.0 and 15ug of pMT4-H516-1D4 construct (controls
did not contain it) in the DMEM+Pen/Strep
3. Then the plates were incubated for 6h
4. The transfection cocktail was removed (without washing) and 2ml of the shock
buffer was added (1x PBS, 10% DMSO, 6mM Dextrose). After 2 min. this shock
buffer was removed. No washing was done after removing the shock buffer. 15 ml
DMEM supplemented with 10% FBS, pen-strep, 0.1mM Chloroquine was added
and cells were incubated 1h.
5. The above medium was removed. Cells were washed with 25ml DMEM. The
cells then were incubated with 30ml of fresh medium (DMEM, 10% FBS, PenStrep) for 48h.
6. Medium was removed, cells were washed with 25 ml ice cold PBS and
harvested in 2ml of ice cold PBS+0.7mM PMSF+0.005% benzamidine by
pipeting.
2) Repair of the RAT mGluR1
On the Fig. 3 the mutations of mGluR1 are shown. Briefly, the strategy of repair is
the following:
First, a PCR reaction (PCR1) with “Start” primer and the primer repairing the
mutation (1) was done, the second PCR (PCR2) with the primer repairing the
mutation (2) and the product of PCR1 as the forward primer was done. The product
of PCR2 was used as the forward primer together with the primer containing
EcoNI site in the third PCR (PCR3). The product of PCR3 was cloned into the
pBlunt vector and sequenced. The plasmids pBlunt+PCR3 and pBlunt+R522 were
cut by EcoNI/MluI and the fragments were ligated. Finally the plasmid pBlunt
containing the Rat mGluR1+StrepTag construct was obtained and sequenced.
Figure 3. Mutations in mGluR1.
Figure 4. Ligation of two mGluR1 fragments.
3) Cloning of H585 and H516 constructs to pMT4 and pACMV-tetO vector.
The H585 and H516 constructs were cut from pBlunt vector by EcoRI and blunted
by Klenow Fragment of DNA Polymerase. The blunted constructs were cloned
into blunted pMT4 and pACMV-tetO vectors the clones were checked for the
orientation of inserts and sequenced.
The following plasmids were obtained:
1) pMT4+H585
2) pMT4+H516
3) pACMV-tetO+H585
4) pACMV-tetO+H516
4) Cloning of H585 and H516 constructs to pMT4 and pACMV-tetO vector.
The mGluR1 construct was cut from pBlunt vector by BamHI and blunted by
Klenow Fragment of DNA Polymerase. The blunted fragment was cloned into
blunted pMT4 and pACMV-tetO vectors. The clones were sequenced.
3. Results
1) Stable Transfection of HEK293 with H516 and H585 constructs
Cells were grown at the following Geneticin concentratrions:
300 µg/ml and 1000 µg/ml
2) The constrcuts H585 and H516 were cloned in pMT4 vector and pACMV-tetO
vector.
3) Transient transfection of COS-1 cells with H516 construct was done, no
expression was observed.
Figure 5. Expression results of H516 construct in COS-1 cells.
Western Blot on His-tag
4. Discussion
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.
Future Work:
1. Checking of HEK293 stably transfected with H516 and H585.
2. Stable transfection of HEK293 with the full-length mGluR1
3. Transient transfection of COS-1 cells with the full-length mGluR1
and H585 construct