PrecisION™ hHCN4-CHO Recombinant Cell Line - Millipore

PrecisION hHCN4-CHO
Recombinant Cell Line
Catalog Number: CYL3012
PRODUCT DESCRIPTION:
Recombinant CHO-K1 cell line expressing the human hyperpolarization-activated, cyclic nucleotide-gated potassium channel 4
(HCN4).
ASSOCIATED PRODUCTS:
The PrecisION hHCN4-CHO Recombinant Cell Line is provided to customers on the purchase of an appropriate license.
The available licenses are:
CYL3012SS PrecisION hHCN4-CHO Single Site License
CYL3012TS PrecisION hHCN4-CHO Two Site License
CYL3012MS PrecisION hHCN4-CHO Multiple Site License
CONTENTS:
2 x 1 mL aliquots containing 1.25 x 10 6 cells/mL in 10% DMSO at passage 14.
STORAGE:
Vials are to be stored in liquid N2.
WARNINGS:
For Research Use Only; Not for Use in Diagnostic Procedures
Not for Animal or Human Consumption
GMO
This product contains genetically modified organisms.
Este producto contiene organismos genéticamente modificados.
Questo prodotto contiene degli organismi geneticamente modificati.
Dieses Produkt enthält genetisch modifizierte Organismen.
Ce produit contient organismes génétiquement des modifiés.
Dit product bevat genetisch gewijzigde organismen.
Tämä tuote sisältää geneettisesti muutettuja organismeja.
Denna produkt innehåller genetiskt ändrade organismer.
MYCOPLASMA TESTING:
The cell line has been screened using the ELISA based Mycoplasma Detection kit (Roche) and by a PCR VenorGem kit (Minerva
Biolabs) to confirm the absence of Mycoplasma species.
FUNCTIONAL VALIDATION:
CHO-K1 cells stably expressing hHCN4 were analysed by IonWorks HT. Over 88% of cells expressed >300 pA outward current
(n=1099) with a mean current amplitude of 1360 pA ± 0.03 (n=971). The HCN4 current was inhibited by standard blockers such as
capsazepine (pIC50 = 4.2 ± 0.1; n=3) and ZD-7288 (pIC50 = 4.4 ± 0.2; n=4).
RECOMMENDED CULTURE CONDITIONS:
Recommended culture conditions and standard operating procedure are provided with the product.
MM09-DJ-CYL3012
DDServices@merckgroup.com ● www.millipore.com/DrugDiscovery

PrecisION hHCN4-CHO
Recombinant Cell Line
Catalog Number: CYL3012
FUNCTIONAL VALIDATION:
Electrophysiological Properties of the hHCN4 current.
Figure 1. IonWorks HT recording from hHCN4-CHO K1 cells.
A. Representative currents evoked from the voltage-command shown in the upper panel. Note the slowly activating inward current
in response to hyperpolarising steps. Calibration bars 2 s and 500 pA.
B. Population histogram for peak (inward) current amplitude at –110 mV obtained from 1099 cells.
MM09-DJ-CYL3012
Count
A
-30mV
B
80
60
40
20
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
I peak at -110mV
-130mV
DDServices@merckgroup.com ● www.millipore.com/DrugDiscovery

PrecisION hHCN4-CHO
Recombinant Cell Line
Catalog Number: CYL3012
Figure 2. Stability of expression over passage (mean peak current for expressors (blue triangles) and % of cells
expressing (red circles)). Each point represents the results from a single IonWorks HT run.
Figure 3. Concentration-response curve and representative current record (inset) for inhibition of hHCN4 currents
by ZD7288 in stably expressing CHO-K1 cells. The abscissa shows the log concentration of ZD7288 (µM) and the
ordinate the current amplitude normalised to the pre-drug value. Each point represents the median value from 2-4
determinations (different cells). From the four parameter logistic equation the IC50 value was 75 µM and the Hill
slope 1.91. hHCN4 currents were evoked by a test step of 4 s from –30 mV to –110 mV. The scale bars in the inset
are 500 ms (x) and 500 pA (y), respectively.
MM09-DJ-CYL3012
Mean Current Amplitude ± SE (nA)
2.5
2.0
1.5
1.0
0.5
0.0
Mean Current (nA)
% Expression
10 12 14 16 18 20 22
Passage Number (Continuous Culture)
% Control Current
100
80
60
40
20
0
+ 300μM
Con
IC 50 value 75μM
Slope 1.91
1 10 100 1000
ZD7288 [μM]
DDServices@merckgroup.com ● www.millipore.com/DrugDiscovery
100
80
60
40
20
0
% Expressing (>0.3 nA)

PrecisION hHCN4-CHO
Recombinant Cell Line
Catalog Number: CYL3012
Vector:
MM09-DJ-CYL3012
XhoI
XbaI
PstI
AhdI
polyA
Neo
KpnI
ScaI
PvuI
pCIN5 hHCN4
8501 bps
Polylinker: CMV-BamHI-NotI-AscI-hHCN4-EcoRI-IRES-neo
hHCN4 Sequence:
The sequence of hHCN4 used to create this stable cell line encodes a protein with the following change to that
encoded by the GenBank accession number AJ238850: Thr110Ser.
Stability of hHCN4-CHO K1 Cell Line:
The hHCN4-CHO K1 cell line has stable expression for >25 passages.
6000
Amp
IRES
ApaI
EcoRI
ApaI
SspI
8000
DDServices@merckgroup.com ● www.millipore.com/DrugDiscovery
4000
MluI
CMV promoter
BamHI
NdeI
SnaBI
PstI
ApaI
2000
hHCN4
KpnI
BamHI
NotI
XhoI
AscI
NotI
PstI
MluI
PstI
PstI
AhdI
ApaI
PstI
KpnI
ScaI

PrecisION hHCN4-CHO
Recombinant Cell Line
Catalog Number: CYL3012
MM09-DJ-CYL3012
For research use only; not for use as a diagnostic.
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our
products are intended for research use only and are not to be used for any other purpose, which includes but is not
limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of
consumption or application to humans or animals.
Certain methods of sample preparation, screening, analysis or use of Millipore products may be covered by
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support any unauthorized or unlicensed use of any third-party owned technology. In particular, Millipore does not
presently possess a license to any patents relating to the use of Millipore products in compound screening.
Purchase of any Millipore product does not imply or grant a license to any such patent to our customers.
©2009: Millipore Corporation. All rights reserved. No part of these works may be reproduced in any form without
permission in writing.
PrecisION is a trademark of Merck KG&A,
Darmstadt, Germany.. IonWorks HT is a
trademark of Molecular Devices Corporation.
DDServices@merckgroup.com ● www.millipore.com/DrugDiscovery