O-064793 GIBCO TET Test.indd - Invitrogen

Tetracycline-Tested GIBCO® FBS
Increase control in tetracycline-induced expression
systems.
Inducible systems allow you to control the expression
level of your gene of interest. Many of the systems currently
available for regulating expression in mammalian
cells rely on tetracycline as the inducing agent, including
Invitrogen’s T-REx System. These systems tend to be very
sensitive, and expression may be activated in the presence
of a minimal amount of tetracycline. Therefore, it is important
that the media and all media components, including
fetal bovine serum (FBS), be free of tetracycline.
How Regulation Works. The T-REx System uses
regulatory elements from the E. coli tetracycline-resistance
operon to manipulate expression from the CMV promoter.
In the absence of tetracycline, the tetracycline repressor
(TR) protein binds the promoter and prevents expression.
The addition of tetracycline causes the TR protein to release
from the promoter and allows expression to proceed (1).
To achieve tightly controlled regulated expression, it is
important that the uninduced system be tetracycline-free.
The lack of tetracycline ensures that expression of the gene
of interest is due only to the addition of defined quantities
of tetracycline to the media. The ideal experiment would
have little or no basal expression of the gene of interest.
Consider the example in which a gene expresses a protein
that produces undesirable effects within the cell (i.e. toxicity).
The unknown presence of tetracycline in the culture
system could potentially cause unwanted expression, leading
to misinterpretation of experimental results.
Table 1. The three criteria of the GIBCO® FBS tetracycline validation assay.
Aspect Description
Linearity
Reproducibility
Stability
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The FBS Factor. Most cell culture systems still employ
supplementation with FBS to promote cell growth. Any
given batch of FBS can contain serum from several thousand
animals. The presence of tetracycline in a batch of serum is
possible when administered to cattle prior to slaughter. As
described earlier, low levels of tetracycline can cause expression
at an undesirable time. While we recommend the addition
of 1 µg/ml tetracycline to induce expression in a T-REx
cell line, the range of 0.1 µg/ml to 1 µg/ml can potentially
induce expression.
The Tet-Tested GIBCO® FBS Solution. To address the
FBS factor, Invitrogen scientists have developed and validated
an assay using reverse-phase HPLC with fluorescence
for detecting the presence of tetracycline in FBS. Using this
assay, as little as 19.7 ng/ml of tetracycline can be detected.
The assay was validated against three criteria: linearity,
reproducibility, and sample stability (Table 1).
GIBCO® Certified and Qualified FBS lots are regularly
tested for tetracycline (Figure 1). When using Invitrogen’s
T-REx System and ordering FBS, you may request a lot
that has been tested for the presence of tetracycline. At the
time of your order, our technical support team will review
the available inventory of the ordered FBS product and
select a lot tested for tetracycline that falls below the limit
of detection. There is no additional charge for this service.
The linear range for the assay is from 50 ng/ml to 1000 ng/ml. A correlation coefficient
of 0.9985 or better is required.
Reproducibility and accuracy at low levels are demonstrated by preparing the low standard
6 times. The %RSD of these samples needs to be 3.93% and 97.8% of the theoretical value.
Spiked samples do not show significant degradation after being freeze-thawed 3 times.
Samples were stored at ambient temperatures unprotected from light for 4 hours.
Quarter 2 2005 Volume 2 Issue 2 QUEST | 29

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To order tetracycline-tested GIBCO® FBS, please visit
www.invitrogen.com/gibco or contact us at locations
worldwide (see page 103). For more information about the
T-REx System, please visit www.invitrogen.com.
Product Quantity Catalog No.
Fetal Bovine Serum, Certified (US) 100 ml 16000-036
500 ml 16000-044
1000 ml 16000-069*
Fetal Bovine Serum, Qualified (non-US) 100 ml 10437-010**
500 ml 10437-028**
1000 ml 10437-036**
Fetal Bovine Serum, Qualified (US) 100 ml 26140-087
500 ml 26140-079
1000 ml 26140-095*
T-REx Complete Kit with pcDNA4/TO© 1 kit K1020-01
T-REx Core Kit with pcDNA4/TO 1 kit K1020-02
T-REx Complete Kit with pcDNA4/TO/myc-His© 1 kit K1030-01
T-REx Core Kit with pcDNA4/TO/myc-His© 1 kit K1030-02
Figure 1. Sample HPLC data from a GIBCO® FBS tetracycline
assay.
A. Tetracycline standard in FBS.
Min
B. Tetracycline screen of a tetra-cycline-tested GIBCO® FBS lot.
8.00
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*This size is custom only
**May not be 7.00 available In Europe
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1. Yao, F. et 4.00 al. (1998) Tetracycline repressor, tetR, rather than the tetR-mammalian
cell transcription factor fusion derivatives, regulates inducible gene expression in
3.00
mammalian cells. Human Gene Therapy 9(13): 1939-1950.
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Tetracycline - 13.414
GIBCO® Certified and Qualified FBS products are routinely
assayed for the presence of tetracycline using reverse-phase
HPLC. A typical screen involves an FBS standard spiked with
tetracycline (A) and a sample from a lot of GIBCO® FBS (B). Note
the absence of the tetracycline spike in the lot being tested.
30 | QUEST Volume 2 Issue 2 Quarter 2 2005
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A. Tetracycline standard in FBS.
Tetracycline - 13.414
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