CD73, also known as 5’-NT, NT5 and NTE, is a glycosyl-phosphatidyl-inositol (GPI) anchored cell membrane protein that belongs to the 5'-nucleotidase (5’-NT) family. The native enzyme exists as a homodimer on the cell surface. CD73 is a purine salvage enzyme expressed on human T and B lymphocytes. CD73 converts extracellular nucleoside­5'­monophosphates to membrane permeable nucleosides, with AMP as the preferred substrate. It also exhibits AMP-, NAD-, and NMN-nucleosidase activities.  CD73 is one of several ectoenzymes responsible for the production of extracellular adenosine, a signaling molecule involved in many physiologic processes, including inflammation, tissue injury response, and cholinergic synapses. CD73 produces extracellular adenosine, which then acts on G protein-coupled purigenic receptors (GPCR) to induce intracellular responses. CD73 is also a regulator of leukocyte extravasation, a function that requires its 5'­nucleotidase activity. CD73 mediates lymphocyte-stromal cell interactions to facilitate lymphocyte development/function.  CD73 has also been considered as a lymphocyte maturation marker independent of its catalytic activity. CD73 serves as a co-stimulatory molecule in activating T cells. The mutation in CD73 gene is associated with calcification of joints and arteries (CALJA), a condition characterized by adult-onset calcification of the lower extremity arteries  and hand and foot capsule joints.

CD73, also known as 5’-NT, NT5 and NTE, is a glycosyl-phosphatidyl-inositol (GPI) anchored cell membrane protein that belongs to the 5'-nucleotidase (5’-NT) family. The native enzyme exists as a homodimer on the cell surface. CD73 is a purine salvage enzyme expressed on human T and B lymphocytes. CD73 converts extracellular nucleoside­5'­monophosphates to membrane permeable nucleosides, with AMP as the preferred substrate. It also exhibits AMP-, NAD-, and NMN-nucleosidase activities.  CD73 is one of several ectoenzymes responsible for the production of extracellular adenosine, a signaling molecule involved in many physiologic processes, including inflammation, tissue injury response, and cholinergic synapses. CD73 produces extracellular adenosine, which then acts on G protein-coupled purigenic receptors (GPCR) to induce intracellular responses. CD73 is also a regulator of leukocyte extravasation, a function that requires its 5'­nucleotidase activity. CD73 mediates lymphocyte-stromal cell interactions to facilitate lymphocyte development/function.  CD73 has also been considered as a lymphocyte maturation marker independent of its catalytic activity. CD73 serves as a co-stimulatory molecule in activating T cells. The mutation in CD73 gene is associated with calcification of joints and arteries (CALJA), a condition characterized by adult-onset calcification of the lower extremity arteries  and hand and foot capsule joints.

 

Product Details

 

Gene Symbol: CD73; 5’-NT; NT; eN; NT5; NTE; eNT; E5NT; CALJA

 

NCBI Gene ID: 4907

 

Uniprot Entry: P21589

 

Construct Details: Full length human CD73 gene is subcloned into the Lentiviral expression vector pLTC with an upstream CMV promoter and a selection marker, which can be used for both transient and stable expression in mammalian cells.  It was co-transfected with the LentiPAK DNA mix (SKU# LP-001) into HEK293 cells to produce high titer lentiviral particles.

 

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

  

Gene Insert Sequence:  

ATGTGTCCCCGAGCCGCGCGGGCGCCCGCGACGCTACTCCTCGCCCTGGGCGCGGTGCTGTGGCCTGCGG

CTGGCGCCTGGGAGCTTACGATTTTGCACACCAACGACGTGCACAGCCGGCTGGAGCAGACCAGCGAGGA

CTCCAGCAAGTGCGTCAACGCCAGCCGCTGCATGGGTGGCGTGGCTCGGCTCTTCACCAAGGTTCAGCAG

ATCCGCCGCGCCGAACCCAACGTGCTGCTGCTGGACGCCGGCGACCAGTACCAGGGCACTATCTGGTTCA

CCGTGTACAAGGGCGCCGAGGTGGCGCACTTCATGAACGCCCTGCGCTACGATGCCATGGCACTGGGAAA

TCATGAATTTGATAATGGTGTGGAAGGACTGATCGAGCCACTCCTCAAAGAGGCCAAATTTCCAATTCTG

AGTGCAAACATTAAAGCAAAGGGGCCACTAGCATCTCAAATATCAGGACTTTATTTGCCATATAAAGTTC

TTCCTGTTGGTGATGAAGTTGTGGGAATCGTTGGATACACTTCCAAAGAAACCCCTTTTCTCTCAAATCC

AGGGACAAATTTAGTGTTTGAAGATGAAATCACTGCATTACAACCTGAAGTAGATAAGTTAAAAACTCTA

AATGTGAACAAAATTATTGCACTGGGACATTCGGGTTTTGAAATGGATAAACTCATCGCTCAGAAAGTGA

GGGGTGTGGACGTCGTGGTGGGAGGACACTCCAACACATTTCTTTACACAGGCAATCCACCTTCCAAAGA

GGTGCCTGCTGGGAAGTACCCATTCATAGTCACTTCTGATGATGGGCGGAAGGTTCCTGTAGTCCAGGCC

TATGCTTTTGGCAAATACCTAGGCTATCTGAAGATCGAGTTTGATGAAAGAGGAAACGTCATCTCTTCCC

ATGGAAATCCCATTCTTCTAAACAGCAGCATTCCTGAAGATCCAAGCATAAAAGCAGACATTAACAAATG

GAGGATAAAATTGGATAATTATTCTACCCAGGAATTAGGGAAAACAATTGTCTATCTGGATGGCTCCTCT

CAATCATGCCGCTTTAGAGAATGCAACATGGGCAACCTGATTTGTGATGCAATGATTAACAACAACCTGA

GACACACGGATGAAATGTTCTGGAACCACGTATCCATGTGCATTTTAAATGGAGGTGGTATCCGGTCGCC

CATTGATGAACGCAACAATGGCACAATTACCTGGGAGAACCTGGCTGCTGTATTGCCCTTTGGAGGCACA

TTTGACCTAGTCCAGTTAAAAGGTTCCACCCTGAAGAAGGCCTTTGAGCATAGCGTGCACCGCTACGGCC

AGTCCACTGGAGAGTTCCTGCAGGTGGGCGGAATCCATGTGGTGTATGATCTTTCCCGAAAACCTGGAGA

CAGAGTAGTCAAATTAGATGTTCTTTGCACCAAGTGTCGAGTGCCCAGTTATGACCCTCTCAAAATGGAC

GAGGTATATAAGGTGATCCTCCCAAACTTCCTGGCCAATGGTGGAGATGGGTTCCAGATGATAAAAGATG

AATTATTAAGACATGACTCTGGTGACCAAGATATCAACGTGGTTTCTACATATATCTCCAAAATGAAAGT

AATTTATCCAGCAGTTGAAGGTCGGATCAAGTTTTCCACAGGAAGTCACTGCCATGGAAGCTTTTCTTTA

ATATTTCTTTCACTTTGGGCAGTGATCTTTGTTTTATACCAATAG

 

Formulation: Pre-packaged lentiviral particles in the conditional medium (serum-free) from HEK293 cells (typical titer 10⁶ - 10⁷ IFU/ml)

 

 

 

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.

 

Transduction Protocol

 

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 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 Target Cell#	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 practices, please refer to NIH’s “Biosafety Considerations for Research with Lentiviral Vectors”.

 

Troubleshooting

 

Poor Transduction Efficiency:

Poor transduction efficiency could be due to a low viral titer. While lentivectors can accommodate relatively large inserts (up to 8 kb) comparing to other systems, there is still a packaging limit. In general, lentivectors could transfer up to 8-kb gene inserts. However, any gene insert larger than 4kb will dramatically decreases packaging efficiency, which would results in a lower viral titer. To overcome low titers, concentrate viral particles or lower target cell density for transduction. If the transduction efficiency is low despite a high titer of virus, the total volume of transduction media on the target cells may be too high. Transduction can be carried out in a volume of media that just covers the cells; this may increase the exposure of the cells to the virus. Low speed spin (e.g., 1500 rpm for 15-30 min) may help increase the likelihood of virus-cell interactions.  Pseudotyping of the transducing virus to target more abundant receptors on a particular cell type is another potential approach.

 

Transduction Kills Target Cells:

It is possible that MOI may be too high. Perform the transduction with a lower MOI or less viral particles. Packaging cell medium may not be compatible for target cell growth. Either dilute the virus in target cell-compatible medium or concentrate and resuspend the virus in medium compatible with the target cell growth. Change the transduction media containing the virus as early as 4 hrs after transduction.

 

 

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

 

Storage

 

The product is shipped at 4°C for immediate use.  Upon receipt, centrifuge the vial briefly before opening.  Store at –80°C or lower and the product is stable for 3 months.  Avoid repeated freeze-thaw cycles.

 

 

The product should be employed in a Biosafety Level 2 tissue culture facility.

 

 

 

 

 

 

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

 

References

 

1.  Cell Signal. 9: 131 (1997)

2. Immunol. Rev. 161:95 (1998).

3. Eur J Immunol. 28: 2981 (1998)

4. J. Biol. Chem. 278:13468 (2003)

5. J Immunol. 180: 4246 (2008)

 

 

 

Documentation

 

Product datasheet (pdf) can be downloaded here: LTV2157-PDS.pdf

 

 

 

Additional supporting documents, including COA and MSDS are available upon request.

 

 

 

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

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 complia