FAQs

pTARGET™ Mammalian Expression Vector System

See Technical Resources for more information.

1. What is the pTARGET™ Vector? Is it based on the pGEM^®-T Vector?
2. Will using the pTARGET™ Vector affect expression levels of my gene of interest?
3. What features does the pTARGET™ Vector include to ensure high levels of expression?
4. Are there any special requirements for protein expression?
5. Which primer is available for sequencing the vector?
6. Can the products of proofreading enzymes, such as Tli DNA Polymerase, be cloned into the pTARGET™ Vector?
7. Can PCR products generated using the Access RT-PCR System be cloned into the pTARGET™ Vector?

1. What is the pTARGET™ Vector? Is it based on the pGEM^®-T Vector?
   The pTARGET™ Mammalian Expression Vector System (Cat.# A1410) is a convenient system for cloning PCR products and expressing cloned PCR products in mammalian cells. The pTARGET™ Vector is based on the pCI-neo Mammalian Expression Vector and is prepared by cutting pCI-neo with EcoRV and adding 3′-T overhangs in a fashion similar to the pGEM^®-T Vector (Cat.# A3610). The principal differences between the pTARGET™ and pCI-neo Mammalian Expression Vectors are the presence of the alpha-peptide coding region of lacZ, which allows blue/white screening on indicator plates, and the single-T overhangs of the cloning site.

2. Will using the pTARGET™ Vector affect expression levels of my gene of interest?
   The pTARGET™ Vector has been designed to ensure that levels of mammalian expression are maximized. Substantial modifications have been made to the alpha peptide of lacZ so that it will not adversely affect expression of the cloned insert. Any potential 5′ splice sites upstream and any 3′ splice sites downstream of the multiple cloning site were removed. Furthermore, sequences that might result in the formation of hairpin structures in the transcribed RNA, which can lead to reduced translation efficiency, were modified to reduce this effect. To confirm the efficacy of these improvements, expression levels of 5 reporter genes using the pTARGET™ Vector were compared to those of the same sequences in the pCI-neo Vector. Expression levels of four of the genes were comparable between the two vectors. Expression levels of the lacZ gene using pTARGET™ were slightly lower; this may be due to interactions between the alpha-peptide sequence and mRNA transcript of the beta-galactosidase gene (1).

3. What features does the pTARGET™ Vector include to ensure high levels of expression?
   The vector includes the CMV immediate-early enhancer/promoter region, SV40 enhancer and early promoter, a chimeric intron downstream of the CMV region and the SV40 late polyadenylation signal. The CMV immediate-early enhancer/promoter region ensures strong, constitutive expression in a variety of cell types. The SV40 early promoter region drives expression of the neomycin phosphotransferase coding region, which can be used to create stable transfectants by G418 selection. The SV40 early promoter region also contains the SV40 origin of replication for induction of transient, episomal replication of the vector in cells that express the SV40 large T antigen such as COS-1 and COS-7 cells. The presence of an intron near the cDNA insert is critical for high expression levels for the majority of cDNA inserts; the exact level of expression enhancement will depend upon the gene that is cloned. The SV40 late polyadenylation signal results in polyadenylation of the transcribed insert RNA, increasing its stability and therefore the steady-state level of RNA resulting in higher protein expression.

4. Are there any special requirements for protein expression?
   An ATG codon for translation initiation MUST be present in the insert DNA and should be the first ATG in the 5′-region. The sequence context in which this codon appears could influence the level of translation (2). Additionally, a stop codon should be present in the expressed sequence since the vector does not encode one.

5. Which primer is available for sequencing the vector?
   The T7 Promoter Primer (Cat.# Q5021) is the only Promega primer that can be used for sequencing. The pUC/M13 primers cannot be used due to the changes in the alpha-peptide coding region.

6. Can the products of proofreading enzymes such as Tli DNA polymerase be cloned into the pTARGET™ Vector?
   Successful ligation into the vector requires that the insert possess single deoxyadenosine overhangs on the 3′-ends. Enzymes that possess 3′->5′ exonuclease activity ("proofreading" activity) produce blunt-end fragments. Single deoxyadenosine overhangs can be added following amplification to produce the required overhangs on the products of proofreading enzymes. Additional dATP and Taq DNA polymerase can be added to the reactions during the final cycles or in a subsequent reaction (3,4).

7. Can PCR products generated using the Access RT-PCR System be cloned into the pTARGET™ Vector?
   The Access RT-PCR System uses Tfl DNA Polymerase, which lacks proofreading activity and produces fragments with A-overhangs. No modifications to the PCR product are required before ligation into the pTARGET™ Vector.

References

1. Brondyk, B. (1996) Promega Notes 58, 2–7.
2. Kozak, M. (1989) J. Cell Biol. 108, 229–41.
3. Kobs, G. (1996) Promega Notes 55, 28–9.
4. Zhou, M.Y., Clark, S.E. and Gomez-Sanchez, C.E. (1995) BioTechniques 19, 34–5.