Human ACE/DCP/CD143 Lentivirus, Full-length Gene in Lentivector, Pre-packaged lentiviral Particles

Description

Angiotensin-converting enzymes including ACE and ACE2 are a central component of the renin-angiotensin system (RAS) that controls blood pressure by regulating the volume of fluids in the body. Both ACE & ACE2 are single-pass type I membrane proteins that belong to the peptidase M2 family.  Unlike ACE2, the extracellular region of ACE contains 2 zinc-binding metallopeptidase domains (40-624aa & 643-1222aa).  ACE catalyzes the conversion of angiotensin I into a physiologically active peptide angiotensin II. Angiotensin II is a potent vasopressor and aldosterone-stimulating peptide that controls blood pressure and fluid-electrolyte balance. ACE also inactivates the vasodilator protein, bradykinin. Accordingly, ACE increases blood pressure and ACE inhibitors are often prescribed to reduce blood pressure. ACE may also play a role in fertility through its ability to cleave and release GPI-anchored membrane proteins in spermatozoa.  Mutations in the ACE gene have been implicated in a wide variety of diseases including cardiovascular conditions, psoriasis, renal disorders, stroke, and Alzheimer's disease. 

 

 

Gene Symbol: ACE; DCP; ACE1; DCP1; CD143

 

NCBI Gene ID: 1636

 

Uniprot Entry: P12821

 

Construct Details: Full length human ACE gene is subcloned into the Lentiviral expression vector pLTC with an upstream CMV promoter and with or without a selection marker, which can be used for both transient and stable expression in mammalian cells.  It can be co-transfected with the LentiPAK DNA mix (SKU# LP-001) into HEK293 cells to produce high titer lentiviral particles. Multiple choices of a stable antibiotic selection marker are available.

 

Vector Type: pLTC or pLTC-IRES-Marker (lentiviral expression vector containing a heterologous CMV promoter +/- a selection marker, see the vector map above)

 

Gene Insert Size: 3921 (bp)

 

Gene Insert Sequence:  

ATGGGGGCCGCCTCGGGCCGCCGGGGGCCGGGGCTGCTGCTGCCGCTGCCGCTGCTGTTGCTGCTGCCGCCGCAGCCCGC

CCTGGCGTTGGACCCCGGGCTGCAGCCCGGCAACTTTTCTGCTGACGAGGCCGGGGCGCAGCTCTTCGCGCAGAGCTACA

ACTCCAGCGCCGAACAGGTGCTGTTCCAGAGCGTGGCCGCCAGCTGGGCGCACGACACCAACATCACCGCGGAGAATGCA

AGGCGCCAGGAGGAAGCAGCCCTGCTCAGCCAGGAGTTTGCGGAGGCCTGGGGCCAGAAGGCCAAGGAGCTGTATGAACC

GATCTGGCAGAACTTCACGGACCCGCAGCTGCGCAGGATCATCGGAGCTGTGCGCACCCTGGGCTCTGCCAACCTGCCCC

TGGCTAAGCGGCAGCAGTACAACGCCCTGCTAAGCAACATGAGCAGGATCTACTCCACCGCCAAGGTCTGCCTCCCCAAC

AAGACTGCCACCTGCTGGTCCCTGGACCCAGATCTCACCAACATCCTGGCTTCCTCGCGAAGCTACGCCATGCTCCTGTT

TGCCTGGGAGGGCTGGCACAACGCTGCGGGCATCCCGCTGAAACCGCTGTACGAGGATTTCACTGCCCTCAGCAATGAAG

CCTACAAGCAGGACGGCTTCACAGACACGGGGGCCTACTGGCGCTCCTGGTACAACTCCCCCACCTTCGAGGACGATCTG

GAACACCTCTACCAACAGCTAGAGCCCCTCTACCTGAACCTCCATGCCTTCGTCCGCCGCGCACTGCATCGCCGATACGG

AGACAGATACATCAACCTCAGGGGACCCATCCCTGCTCATCTGCTGGGAGACATGTGGGCCCAGAGCTGGGAAAACATCT

ACGACATGGTGGTGCCTTTCCCAGACAAGCCCAACCTCGATGTCACCAGTACTATGCTGCAGCAGGGCTGGAACGCCACG

CACATGTTCCGGGTGGCAGAGGAGTTCTTCACCTCCCTGGAGCTCTCCCCCATGCCTCCCGAGTTCTGGGAAGGGTCGAT

GCTGGAGAAGCCGGCCGACGGGCGGGAAGTGGTGTGCCACGCCTCGGCTTGGGACTTCTACAACAGGAAAGACTTCAGGA

TCAAGCAGTGCACACGGGTCACGATGGACCAGCTCTCCACAGTGCACCATGAGATGGGCCATATACAGTACTACCTGCAG

TACAAGGATCTGCCCGTCTCCCTGCGTCGGGGGGCCAACCCCGGCTTCCATGAGGCCATTGGGGACGTGCTGGCGCTCTC

GGTCTCCACTCCTGAACATCTGCACAAAATCGGCCTGCTGGACCGTGTCACCAATGACACGGAAAGTGACATCAATTACT

TGCTAAAAATGGCACTGGAAAAAATTGCCTTCCTGCCCTTTGGCTACTTGGTGGACCAGTGGCGCTGGGGGGTCTTTAGT

GGGCGTACCCCCCCTTCCCGCTACAACTTCGACTGGTGGTATCTTCGAACCAAGTATCAGGGGATCTGTCCTCCTGTTAC

CCGAAACGAAACCCACTTTGATGCTGGAGCTAAGTTTCATGTTCCAAATGTGACACCATACATCAGGTACTTTGTGAGTT

TTGTCCTGCAGTTCCAGTTCCATGAAGCCCTGTGCAAGGAGGCAGGCTATGAGGGCCCACTGCACCAGTGTGACATCTAC

CGGTCCACCAAGGCAGGGGCCAAGCTCCGGAAGGTGCTGCAGGCTGGCTCCTCCAGGCCCTGGCAGGAGGTGCTGAAGGA

CATGGTCGGCTTAGATGCCCTGGATGCCCAGCCGCTGCTCAAGTACTTCCAGCCAGTCACCCAGTGGCTGCAGGAGCAGA

ACCAGCAGAACGGCGAGGTCCTGGGCTGGCCCGAGTACCAGTGGCACCCGCCGTTGCCTGACAACTACCCGGAGGGCATA

GACCTGGTGACTGATGAGGCTGAGGCCAGCAAGTTTGTGGAGGAATATGACCGGACATCCCAGGTGGTGTGGAACGAGTA

TGCCGAGGCCAACTGGAACTACAACACCAACATCACCACAGAGACCAGCAAGATTCTGCTGCAGAAGAACATGCAAATAG

CCAACCACACCCTGAAGTACGGCACCCAGGCCAGGAAGTTTGATGTGAACCAGTTGCAGAACACCACTATCAAGCGGATC

ATAAAGAAGGTTCAGGACCTAGAACGGGCAGCACTGCCTGCCCAGGAGCTGGAGGAGTACAACAAGATCCTGTTGGATAT

GGAAACCACCTACAGCGTGGCCACTGTGTGCCACCCGAATGGCAGCTGCCTGCAGCTCGAGCCAGATCTGACGAATGTGA

TGGCCACGTCCCGGAAATATGAAGACCTGTTATGGGCATGGGAGGGCTGGCGAGACAAGGCGGGGAGAGCCATCCTCCAG

TTTTACCCGAAATACGTGGAACTCATCAACCAGGCTGCCCGGCTCAATGGCTATGTAGATGCAGGGGACTCGTGGAGGTC

TATGTACGAGACACCATCCCTGGAGCAAGACCTGGAGCGGCTCTTCCAGGAGCTGCAGCCACTCTACCTCAACCTGCATG

CCTACGTGCGCCGGGCCCTGCACCGTCACTACGGGGCCCAGCACATCAACCTGGAGGGGCCCATTCCTGCTCACCTGCTG

GGGAACATGTGGGCGCAGACCTGGTCCAACATCTATGACTTGGTGGTGCCCTTCCCTTCAGCCCCCTCGATGGACACCAC

AGAGGCTATGCTAAAGCAGGGCTGGACGCCCAGGAGGATGTTTAAGGAGGCTGATGATTTCTTCACCTCCCTGGGGCTGC

TGCCCGTGCCTCCTGAGTTCTGGAACAAGTCGATGCTGGAGAAGCCAACCGACGGGCGGGAGGTGGTCTGCCACGCCTCG

GCCTGGGACTTCTACAACGGCAAGGACTTCCGGATCAAGCAGTGCACCACCGTGAACTTGGAGGACCTGGTGGTGGCCCA

CCACGAAATGGGCCACATCCAGTATTTCATGCAGTACAAAGACTTACCTGTGGCCTTGAGGGAGGGTGCCAACCCCGGCT

TCCATGAGGCCATTGGGGACGTGCTAGCCCTCTCAGTGTCTACGCCCAAGCACCTGCACAGTCTCAACCTGCTGAGCAGT

GAGGGTGGCAGCGACGAGCATGACATCAACTTTCTGATGAAGATGGCCCTTGACAAGATCGCCTTTATCCCCTTCAGCTA

CCTCGTCGATCAGTGGCGCTGGAGGGTATTTGATGGAAGCATCACCAAGGAGAACTATAACCAGGAGTGGTGGAGCCTCA

GGCTGAAGTACCAGGGCCTCTGCCCCCCAGTGCCCAGGACTCAAGGTGACTTTGACCCAGGGGCCAAGTTCCACATTCCT

TCTAGCGTGCCTTACATCAGGTACTTTGTCAGCTTCATCATCCAGTTCCAGTTCCACGAGGCACTGTGCCAGGCAGCTGG

CCACACGGGCCCCCTGCACAAGTGTGACATCTACCAGTCCAAGGAGGCCGGGCAGCGCCTGGCGACCGCCATGAAGCTGG

GCTTCAGTAGGCCGTGGCCGGAAGCCATGCAGCTGATCACGGGCCAGCCCAACATGAGCGCCTCGGCCATGTTGAGCTAC

TTCAAGCCGCTGCTGGACTGGCTCCGCACGGAGAACGAGCTGCATGGGGAGAAGCTGGGCTGGCCGCAGTACAACTGGAC

GCCGAACTCCGCTCGCTCAGAAGGGCCCCTCCCAGACAGCGGCCGCGTCAGCTTCCTGGGCCTGGACCTGGATGCGCAGC

AGGCCCGCGTGGGCCAGTGGCTGCTGCTCTTCCTGGGCATCGCCCTGCTGGTAGCCACCCTGGGCCTCAGCCAGCGGCTC

TTCAGCATCCGCCACCGCAGCCTCCACCGGCACTCCCACGGGCCCCAGTTCGGCTCCGAGGTGGAGCTGAGACACTCCTG

A

 

Formulation: Lentivector encoded and pre-packaged viral particles (typical titer 10⁶ - 10⁷ IFU/ml) in the conditional medium (serum-free) from HEK293 cells that have been transfected with the lentivector gene clone and the LentiPAK DNA mix

 

 

 

FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMAN.

Important Safety Information: With the safety features in place, our lentiviral vectors and viral particles can be employed in standard Biosafety Level 2 tissue culture facilities and should be treated with the same level of caution as any other potentially infectious agent. Any investigator who purchases our lentiviral/retroviral products & services is responsible for following Biosafety Level 2 requirements on the handling of viral particles. For more information on Biosafety Level 2 agents and practices, please refer to NIH’s “Biosafety Considerations for Research with Lentiviral Vectors”.

Restriction: This product is not transferable or re-sellable.  Customer obtain no right to transfer, assign, or sublicense its use rights, or to transfer, resell, package, or otherwise distribute the product, or to use the product for the benefit of any third party or for any commercial purpose.  Customer may only use the product in compliance with applicable local, state and federal laws, regulations and rules.  Customer may not directly or indirectly use the product or allow the transfer, transmission, export or re-export of all or any part of the product in violation of any export control law or regulation of the united states or any other relevant jurisdiction.  Your use of this product constitutes acceptance of the terms of this limited use agreement.  Please refer to our “terms & conditions” for details.  If you are not willing to accept the limitation of this agreement, G&P Biosciences will accept return of the product for a full/partial refund.

 

 

Pre-packaged lentiviral particles are most advanced gene delivery tools. Each particles contain a fully sequence verified target gene and are psudotyped with the VSV-G glycoprotein, ready for transduction into into a wide range of cell types including hard-to-transfect primary cells and non-dividing cells. They are supplied in 1-mL aliquot(s) of the serum-/antibiotic-free solution suitable for both in vitro and in vivo delivery. They are produced in HEK293 packaging cells with a typical titer of ≥10⁷ IFU/ml using our optimized LentPAK^TM packaging system. The lentiviral particles can be used to transduce subconfluent target cells. Depending on your purpose, you may choose a specific multiplicity of infection (MOI) or test a range of MOIs to determine which gives you the desired results. Transduction can be enhanced by the addition of polybrene, also known as hexadimethrine bromide (typically at 8-10 μg/ml). 

 

Quick Protocol for Transduction

 

Day 1. Seeding Target Cells

For an example, plate target cells in a 10 cm plate at a density of 1 - 5x 10⁵ cells/ml that will produce approximately 60% confluency in 24 hours.

 

Note: other size plates can also be used depending on the nature of your experiment. Adjust the reagent amount according to Table 1

 

Table 1. Seeding Density of Target Cells (1 day prior to transduction)

Vessel Type	Seeding Density	Volume of Media
10-cm dish	1 – 5 x 106	10 mL
6-well plate	0.3 – 1 x 106	2 mL/well
12-well plate	0.15 – 0.5 x 106	1 mL/well
24-well plate	0.6 – 2 x 105	0.5 mL/well
96-well plate	1 – 4 x 104	0.1 mL/well

  

Day 2. Transduction

Remove the growth media from the dish/plate prepared the day before. Replace with 1/2 volume of culture medium containing desired amount of lentiviral particles (at 2 to 20 MOI).  For example, add 4.5 mL of growth medium and 0.5 mL of Lentiviral particles, or simply add 5 mL of Lentiviral particles (for a low titer viral preparation or a high MOI transduction). Add polybrene directly to the media on the target cells at 8 μg/ml. Mix by gentle swirling.

 

Incubate at 37°C with 5% CO2 for 4 - 24 hours, then replace the transduction medium with right amount of growth medium according to table 1 (for example 10 mL for 10-cm dish). Culture the cells for 48 – 72 hours, and transduced cells are ready for downstream analyses.

 

Note: Adjust volumes accordingly for transduction of other plate types.  For example, add 1 ml of growth medium and lentiviral particle mixture for 6-well plate, 0.5 ml for 12-well and 0.25 mL for 24-well except for 96 well, in which 100 μl should be used. The change of transduction medium is often unnecessary with our pre-packaged lentiviral particles.  Simply culture cells for 3-4 days before analysis. 

 

The virus-containing media can be changed in as short as 4 hours after transduction if toxicity of the lentiviral transduction is a concern. Normally reverse transcription and genome integration of the lentivector takes place within 24-36 hours. With our ready-to-use, prepackaged lentiviral particles, the change of media is often unnecessary. The transduction process can be ongoing for 2 - 6 days without significant impact on cell growth/viability. The transduction process can also be repeated if desired. For a lentivector with a fluorescent reporter (such as GFP), FACS can be used to enrich for cells that express GFP. If it contains a drug selectable marker, follow the protocol for the particular drug. For example, puromycin selection is usually carried out at 1-10 μg/mL, depending on the target cells’ sensitivity

 

 

Important Safety Information

With the safety features in place, our lentiviral vectors and viral particles can be employed in standard Biosafety Level 2 tissue culture facilities and should be treated with the same level of caution as any other potentially infectious agent. Any investigator who purchases our lentiviral/retroviral products & services is responsible for following Biosafety Level 2 requirements on the handling of viral particles. For more information on Biosafety Level 2 agents and&