pc DNA3

Special features and applications

1.   Topic: pcDNA and CMV By Dr. Vijay Kumar Assistant Professor Department of Biosciences, Swami Rama Himalayan University

2. Learning objectives At the end of this learning session a student of  M .Sc. Microbiology/Biotechnology –Semester-IV should be able to: Expression vectors- pcDNA & CMV Characteristics Examples

3. pcDNA3.1(+) and pcDNA3.1(-) are 5.4 kb vectors derived from pcDNA3 and designed for high-level stable and transient expression in mammalian hosts. High-level stable and non-replicative transient expression can be carried out in most mammalian cells. The vectors contain the following elements: Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cells Multiple cloning sites in the forward (+) and reverse (-) orientations to facilitate cloning Neomycin resistance gene for selection of stable cell lines Episomal replication in cells lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g. COS-1, COS-7) The control plasmid, pcDNA3.1/CAT, is included for use as a positive control for transfection and expression in the cell line of choice pcDNA3.1(+) pcDNA3.1(-)

4. pcDNA3.1/CAT pcDNA3.1/CAT is a 6217 bp control vector containing the gene for CAT. It was constructed by digesting pcDNA3.1(+) with Xho I and Xba I and treating with Klenow . An 800 bp Hind III fragment containing the CAT gene was treated with Klenow and then ligated into pcDNA3.1(+)

5. pcDNA™3.1/ myc -His(-) A, B, and C pcDNA™3.1/ myc -His(-) A, B, and C are 5.5 kb vectors designed for overproduction of recombinant proteins in mammalian cell lines. The vectors are supplied in three reading frames to facilitate in frame cloning, with a C-terminal peptide containing a polyhistidine metal-binding tag and the myc (c- myc ) epitope. The human cytomegalovirus immediate-early (CMV) promoter provides high level expression in a wide range of mammalian cells. In addition, the vector will replicate episomally in cell lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g. COS7). High-level stable and nonreplicative transient expression can be carried out in most mammalian cells. The control plasmid, pcDNA™3.1/ myc -His(-)/ lacZ , is the pcDNA™3.1/ myc -His(-) A vector with a 3.2 kb fragment containing the β-galactosidase gene cloned in frame with the C-terminal peptide It is included for use as a positive control for transfection, expression, and detection in the cell line of choice.

6. C ytomegalovirus expression system The strong human cytomegalovirus (CMV) promoter regulatory region drives constitutive protein expression levels as high as 1 mg/L in COS cells. For less potent cell lines, protein levels are typically ~0.1 mg/L. The presence of the SV40 replication origin will result in high levels of DNA replication in SV40 replication permissive COS cells. CMV vectors contain the pMB1 (derivative of pBR322) origin for replication in bacterial cells, the b-lactamase gene for ampicillin resistance selection in bacteria, hGH polyA , and the f1 origin. Vectors containing the preprotrypsin leader (PPT) sequence direct secretion of FLAG fusion proteins into the culture medium for purification using ANTI-FLAG® antibodies, resins, and plates.

7. Transient Expression CMV vectors for high-level transient expression offer a number of fusion tag formats (standard FLAG® and 3xFLAG® tags as well as dual tags such as FLAG- Myc , 3xFLAG-Myc, and FLAG-MAT™) as shown below. The recognition sequence for enterokinase , Asp-Asp-Asp-Asp-Lys, is found at the C-terminal end of the FLAG® epitope tag. Removal of FLAG is possible in all fusion proteins containing an N-terminal FLAG sequence. Dual tag fusion proteins may also be cleaved with enterokinase for removal of one or more tags, depending on the position of FLAG in the protein sequence. CMV vectors contain the pMB1 (derivative of pBR322) origin for replication in bacterial cells, the b-lactamase gene for ampicillin resistance selection in bacteria, hGH polyA , and the f1 origin. Vectors containing the preprotrypsin leader (PPT) sequence direct secretion of FLAG fusion proteins into the culture medium for purification using ANTI-FLAG® antibodies, resins, and plates

8. Stable Expression Our vectors for stable expression, as those for transient expression, contain the strong CMV promoter for high-level constitutive expression in mammalian cells. In addition, these constructs carry the aminoglycoside phosphotransferase II gene (neomycin resistance gene or neor ) that confers resistance to aminoglycosides such as G 418 sulfate, allowing selection of stable transfectants . CMV vectors also contain the pMB1 (derivative of pBR322) origin for replication in bacterial cells, the b-lactamase gene for ampicillin resistance selection in bacteria, hGH polyA , and the f1 origin. Vectors containing the preprotrypsin leader (PPT) sequence direct secretion of FLAG fusion proteins into the culture medium for purification using ANTI-FLAG® antibodies, resins, and plates.

9. pCMV6 Mammalian Expression Vectors– Physical Maps of pCMV6-XL4 , pCMV6-XL5 and pCMV6-XL6

10. Description of the vectors The pCMV6 series of vectors were used in the construction of all OriGene Rapid-Screen libraries and therefore are the vectors present in all TrueClones . Therefore, these “empty” vectors are the most appropriate negative control plasmids for use in any over-expression assay of TrueClones . All three vectors contain the same polylinker (Sac I to Sma I). The CMV promoter, which can be used to express the cloned cDNA, is followed by the hGH (human growth hormone) polyA signal located downstream of the insert. The ColE1 ori is the bacterial origin of replication, the SV40 ori allows for replication in mammalian cells and the f1 ori is the filamentous phage origin of replication, which allows for the recovery of single-stranded plasmids. The ampicillin resistant gene confers the selection of the plasmid in E. coli.

11. Mammalian Cell Protein Over-Expression: The polylinker sites are downstream of a CMV promoter * capable of driving heterologous gene expression in a variety of mammalian cell lines in culture. However, there are examples in the literature suggesting that post-transcriptional and/or regulation may affect the achievable protein expression levels in a given cell line. OriGene recommends the use of COS cells or HEK293 cells, however, the transfection conditions for the cell line as well as the assay conditions for the protein must first be optimized .

12. In vitro Transcription: T7 RNA polymerase can be used for generating transcripts of the cDNA by in vitro transcription. However, the pCMVXL4 vector has a second but opposing T7 site (with a one-base substitution on the 3’ end) located between ColE1 ori and SV40 ori . Inserts (cDNA) in this vector must first be released by digesting with Sac I and Sma I before the in vitro transcription reaction. The pCMV-XL5 vector does not have a second T7 promoter site, and the pCMV-XL6 vector has an SP6 promoter. Therefore, clones within the XL5 or XL6 vectors do not require prior insert excision. The M13 primer sequence is represented twice within the pCMV6-XL4 and pCMV6-XL5 vector sequences with only a slight variation in bases between the two sites. Therefore, sequencing from the 3’ end of cDNA inserts may not be performed using an M13 Reverse primer.

13. Suggested Readings Sambrook , J., Russell, D. W., & Russell, D. W. (2008). Molecular cloning, a laboratory manual (3-volume set) Cold spring harbor laboratory press Cold Spring Harbor , New York (2001). Sterer , N., Hendler , A., Davidi , MP & Rosenberg, M. A novel microscopic assay for oral malodour-related microorganisms.  J Breath Res ,  2 , 026003 . Gruh , I., Wunderlich , S., Winkler, M., Schwanke , K., Heinke , J., Blömer , U., ... & Martin, U. (2008). Human CMV immediate‐early enhancer: a useful tool to enhance cell‐type‐specific expression from lentiviral vectors.  The Journal of Gene Medicine: A cross‐disciplinary journal for research on the science of gene transfer and its clinical applications ,  10 (1), 21-32. Boulos , S., Meloni , B. P., Arthur, P. G., Bojarski , C., & Knuckey , N. W. (2006). Assessment of CMV, RSV and SYN1 promoters and the woodchuck post-transcriptional regulatory element in adenovirus vectors for transgene expression in cortical neuronal cultures.  Brain research ,  1102 (1), 27-38.