T-REx™ Core Kit, with pcDNA™4/TO Vector - FAQs

查看更多产品信息 T-REx™ Core Kit, with pcDNA™4/TO Vector - FAQs (K102002)

4 个常见问题解答

What dose of tetracycline or doxycycline inducer should I administer to transgenic mice harboring the T-REx system or other tet-inducible system?

We have not performed such experiments at Thermo Fisher Scientific, but external references indicate that you may use doxycycline or tetracycline at 200µg/ml in drinking water. Add 5% sucrose to the drinking water - the sugar makes the water palatable for the mice.

Specific citations for these methods: Immunity 2000, 12(5):459-469; PNAS 1996, 93:10933-10938; Eur J Biochem 1999, 260(2):533-539.

What concentration of doxycycline should I use for induction in the T-REx system?

The recommended concentration of doxycycline for induction in the T-REx system can vary from 10ng/ml -1µg/ml. For best results, you should pre-test the dose response for your specific cell line.

Is the sequence of the CMV promoter in pcDNA3.1 vector complete, or is it only the core CMV promoter?

In addition to the CAAT and TATA boxes, the CMV promoter in pcDNA3.1 vector contains sequence homologous to base pairs 137 to 724 of the sequence submitted by Boshart, et al (GenBank Accession # K03104). The complete enhancer region is contained between 214 and 620 of this sequence. Therefore, by this definition, the CMV promoter could be said to be "complete".

Please note that this promoter does not contain an intron. Some people believe that the complete promoter must contain the intron, but that has not been demonstrated to be necessary for expression.

Does the BGH poly A region in your vectors have a splice donor and splice acceptor (and therefore an intron)? What is the general function of the BGH polyA region?

There is no intron in the BGH polyA sequence. The BGH polyA signal (bases 1028-1252 in pcDNA3.1 vector) is the sequence that allows for polyadenylation of the RNA transcript. It is described in the following reference: The 3'-flanking sequence of the bovine growth hormone gene contains novel elements required for efficient and accurate polyadenylation. J Biol Chem.1992 Aug 15;267(23):16330-4

The reference states that the 3' untranslated region leading up to the poly adenylation core sequence (AATAAA) contains unique disperse non-consensus elements (consensus elements are poly U and GU-rich tracts) that serve to increase the efficiency of polyadenylation to a high degree, and that a 9-base sequence downstream of the core sequence facilitates efficient cleavage of the RNA strand. This leads to more stable and higher-abundance transcripts.