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pTracer™-SV40 Mammalian Expression Kit - FAQs

View additional product information for pTracer™-SV40 Mammalian Expression Kit - FAQs (V87001)

3 product FAQs found

After stably transfecting pTracer-SV40 vector into HL60 cells with Zeocin antibiotic selection at 50 ug/mL, 60% to 70% of the cells were observed to express GFP at the end of 4 weeks. However at the end of 6 weeks, only 1% to 2% of the cells now express GFP. Why has the GFP expression decreased?

Here are some possible reasons for this observation:
1. The Zeocin antibiotic concentration in this case appears to be low. It is best to perform a kill curve to determine the appropriate concentration.
2. It is possible that Zeocin antibiotic resistant populations, that do not express large amounts of GFP, are now dominating the growth of this culture.
3. It is also possible that a Zeocin antibiotic resistant population with no expression of the Zeo-GFP is dominating the culture. Some cell types (CHO is a good example) have very good MDR (multi-drug resistance) membrane proteins that pump out the drug as fast as it comes in. They make themselves resistant without actually expressing the Zeocin antibiotic resistance gene. The low concentration of Zeocin antibiotic could select for these drug resistant cells. Addition of a higher Zeocin antibiotic concentration may fix this experiment, although once a cell has upregulated its MDR genes, it is often hard to kill.

We recommend that the experiment be performed again using a higher concentration of Zeocin antibiotic during selection (e.g. 100 -200 µg/ml).

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.