pVITRO2-GFP/Lacz

Expression plasmids pVITRO2-GFP/Lacz are developed mainly for in vitro studies.

pVITRO2-GFP/Lacz allows the ubiquitous and constitutive co-expression of two reporter genes: GFP (green fluorescent protein) and LacZ (β-galactosidase).

pVITRO2-GFP/Lacz plasmids can be stably transfected in mammalian cells and the reporter genes are expressed at high levels.

pVITRO2-GFP/Lacz plasmids carry two human ferritin composite promoters, FerH (heavy chain) and FerL (light chain). To eliminate the iron regulation, their 5’UTRs have been replaced by the 5’UTR of the mouse and chimpanzee EF-1α genes.

pVITRO2-GFP/Lacz plasmids are available with different selectable markers that are active both in E. coli and mammalian cells.

Specifications

Promoters: SV40/hFerH/mEF1α & CMV/hFerL/chEF1α

Selection markers active both in E. coli and mammalian cells:

•   bsr (blasticidin resistance)
•   hph (hygromycin resistance)
•   neo (kanamycin/G418 resistance).

Reporter Genes:

•   GFP (green fluorescent protein)
•   LacZ (β-galactosidase)

Contents

pVITRO2-neo-GFP/LacZ

• 20 µg of lyophilized DNA

pVITRO2-hygro-GFP/LacZ

• 20 µg of lyophilized DNA
• 1 ml of Hygromycin B Gold (hygromycin) at 100 mg/ml

pVITRO2-blasti-GFP/LacZ

• 20 µg of lyophilized DNA
• 2 x 1 ml blasticidin at 10 mg/ml

Product is shipped at room temperature

Description

Gene Combinations in a Single Plasmid

Co-expression of two or more genes from a single vector is more efficient and convenient than using two separate vectors. pVITRO2-GFP/LacZ are multigenic plasmids that contain two distinct transcription units (TU).

Strong and Constitutive Expression of Two Transgenes

Each pVITRO2-GFP/LacZ plasmid features two constitutive promoters that drive high levels of expression from two separate TU in a large number of mammalian cell lines.
Transcriptional interference is minimized by using promoters that are coordinately activated, and strong polyadenylation signals (polyA). pVITRO2-GFP/LacZ plasmids contain two human ferritin composite promoters, FerH (heavy chain) and FerL (light chain), combined to the SV40 and CMV enhancers respectively. To eliminate the iron regulation, their 5’UTRs have been replaced by the 5’UTR of the mouse and chimpanzee EF-1α genes.

Single Selection Marker for E. coli and Mammalian Cells

pVITRO2-GFP/LacZ plasmids are available with a selectable marker that is active both in E. coli and mammalian cells: bsr (blasticidin resistance), hph (hygromycin resistance) or neo (kanamycin/G418 resistance).
In bacteria, the resistance gene is expressed from the E. coli EM7 promoter. In mammalian cells, it is transcribed from the promoter located 3’ of the Ori, as a polycistronic mRNA and translated through the IRES of the Foot and Mouth Disease Virus.

Two Reporter Genes

pVITRO2-GFP/LacZ express two reporter genes: GFP and LacZ
GFP (green fluorescent protein) and LacZ (β-galactosidase) are commonly used reporter proteins.
The E. coli lacZ gene codes for the enzyme β-galactosidase which catalyzes the hydrolysis of the substrate X-Gal to produce a blue color that is easily visualized under a microscope.
The red-shifted variant of the jellyfish GFP gene encodes a green fluorescent protein that absorbs blue light (major peak at 480 nm) and emits green light (major peak at 505 nm).

pVITRO2-GFP/LacZ plasmids can be used as control plasmids for pVITRO2-MCS or cloning vectors. The reporter genes are flanked by unique restriction sites and can be easily replaced by open reading frames chosen from InvivoGen’s Gene A-List.

Details

• hFerH and hFerL composite promoters: Ferritin is a 24 subunit protein composed of two subunit types, termed H (heavy) and L (light). Ferritin is ubiquitously expressed. Its synthesis is highly regulated by the iron status of the cell. To eliminate the iron regulation of the ferritin promoters, the 5’UTR of FerH and FerL have been replaced by the 5’UTR of the mouse and chimpanzee elongation factor 1 (EF1) genes, respectively.

• SV40 enhancer which is comprised of a 72-base-pair repeat allows the enhancement of gene expression in a large host range. The enhancement varies from 2-fold in non-permissive cells to 20-fold in permissive cells.

• CMV enhancer: The major immediate early enhancer of the human cytomegalovirus (HCMV) is composed of unique and repeated sequence motifs. The HCMV enhancer can substitute for the 72-bp repeats of SV40 and is severalfold more active than the SV40 enhancer.

• SV40 pAn: the Simian Virus 40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mRNA.

• pMB1 ori: a minimal E. coli origin of replication to limit vector size, but with the same activity as the longer Ori.

• FMDV IRES: The internal ribosome entry site of the Foot and Mouth Disease Virus enables the translation of two open reading frames from one mRNA with high levels of expression.

• EM7 is a bacterial promoter that enables the constitutive expression of the antibiotic resistance gene in E. coli.

•  Antibiotic resistance gene :
- Resistance to Blasticidin S is conferred by the bsr gene from Bacillus cereus.
- hph gene confers resistance to Hygromycin B both in E. coli and mammalin cells.
- The neo gene from Tn5 confers resistance to Kanamycin in E.coli and G418 in mammalian cells.
In bacteria, the resistance gene is expressed from the constitutive E. coli EM7 promoter. In mammalian cells, the resistance gene is transcribed from the rat EF-1α promoter as a polycistronic mRNA and translated via the FMDV IRES.

• EF1 pAn is a strong polyadenylation signal.

• LacZ gene: The E. coli lacZ gene codes for the enzyme β-galactosidase which catalyzes the hydrolysis of the substrate X-Gal to produce a blue color that is easily visualized under a microscope.

• GFP gene: This red-shifted variant of the jellyfish GFP gene encodes a green fluorescent protein that absorbs blue light (major peak at 480 nm) and emits green light (major peak at 505 nm).