CD6, also known as TP120, is a single-pass type I membrane protein of the group B scavenger receptor cysteine-rich (SRCR) superfamily. CD6 contains three SRCR domains in the extracellular region. CD6 is a cell surface glycoprotein expressed primarily on T cells and thus a T-cell differentiation antigen. CD6 is expressed by thymocytes, mature T-cells, a subset of B-cells known as B-1 cells, and by some neuronal cells in the brain.  CD6 may function as both a co-stimulatory molecule in T cell activation and as an adhesion receptor.  As a cell adhesion molecule, CD6 mediates cell-cell contacts and regulates T cell responses via its interaction with the ligand ALCAM (activated leukocyte cell adhesion molecule) or CD166. CD6 partially associates with the TCR/CD3 complex and colocalizes with it at the center of the mature immunological synapse on T lymphocytes. CD6/ALCAM interactions are thought to play a role in thymocyte development.  CD6 may also contribute to signaling cascades triggered by activation of the TCR/CD3 complex and the overall functional avidity of selection. CD6 also functions as calcium-dependent pattern receptor that binds and aggregates both Gram-positive and Gram-negative bacteria. CD6 binds both lipopolysaccharide (LPS) from Gram-negative bacteria and lipoteichoic acid from Gram-positive bacteria. CD6 thus mediates activation of the inflammatory response and the secretion of pro-inflammatory cytokines in response to LPS. CD6 may be associated with susceptibility to multiple sclerosis

CD6, also known as TP120, is a single-pass type I membrane protein of the group B scavenger receptor cysteine-rich (SRCR) superfamily. CD6 contains three SRCR domains in the extracellular region. CD6 is a cell surface glycoprotein expressed primarily on T cells and thus a T-cell differentiation antigen. CD6 is expressed by thymocytes, mature T-cells, a subset of B-cells known as B-1 cells, and by some neuronal cells in the brain.  CD6 may function as both a co-stimulatory molecule in T cell activation and as an adhesion receptor.  As a cell adhesion molecule, CD6 mediates cell-cell contacts and regulates T cell responses via its interaction with the ligand ALCAM (activated leukocyte cell adhesion molecule) or CD166. CD6 partially associates with the TCR/CD3 complex and colocalizes with it at the center of the mature immunological synapse on T lymphocytes. CD6/ALCAM interactions are thought to play a role in thymocyte development.  CD6 may also contribute to signaling cascades triggered by activation of the TCR/CD3 complex and the overall functional avidity of selection. CD6 also functions as calcium-dependent pattern receptor that binds and aggregates both Gram-positive and Gram-negative bacteria. CD6 binds both lipopolysaccharide (LPS) from Gram-negative bacteria and lipoteichoic acid from Gram-positive bacteria. CD6 thus mediates activation of the inflammatory response and the secretion of pro-inflammatory cytokines in response to LPS. CD6 may be associated with susceptibility to multiple sclerosis

 

Product Details

 

Gene Symbol: CD6; TP120; T12

 

NCBI Gene ID: 923

 

Uniprot Entry: P30203

 

Construct Details: Full length human CD6 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, see the vector map above)

 

Gene Insert Sequence:  

ATGTGGCTCTTCTTCGGGATCACTGGATTGCTGACGGCAGCCCTCTCAGGTCATCCATCTCCAGCCCCAC

CTGACCAGCTCAACACCAGCAGTGCAGAGAGTGAGCTCTGGGAGCCAGGGGAGCGGCTTCCGGTCCGTCT

GACAAACGGGAGCAGCAGCTGCAGCGGGACGGTGGAGGTGCGGCTCGAGGCGTCCTGGGAGCCCGCGTGC

GGGGCGCTCTGGGACAGCCGCGCCGCCGAGGCCGTGTGCCGAGCACTGGGCTGCGGCGGGGCGGAGGCCG

CCTCTCAGCTCGCCCCGCCGACCCCTGAGCTGCCGCCCCCGCCTGCAGCCGGGAACACCAGCGTAGCAGC

TAATGCCACTCTGGCCGGGGCGCCCGCCCTCCTGTGCAGCGGCGCCGAGTGGCGGCTCTGCGAGGTGGTG

GAGCACGCGTGCCGCAGCGACGGGAGGCGGGCCCGTGTCACCTGTGCAGAGAACCGCGCGCTGCGCCTGG

TGGACGGTGGCGGCGCCTGCGCCGGCCGCGTGGAGATGCTGGAGCATGGCGAGTGGGGATCAGTGTGCGA

TGACACTTGGGACCTGGAGGACGCCCACGTGGTGTGCAGGCAACTGGGCTGCGGCTGGGCAGTCCAGGCC

CTGCCCGGCTTGCACTTCACGCCCGGCCGCGGGCCTATCCACCGGGACCAGGTGAACTGCTCGGGGGCCG

AAGCTTACCTGTGGGACTGCCCGGGGCTGCCAGGACAGCACTACTGCGGCCACAAAGAGGACGCGGGCGC

GGTGTGCTCAGAGCACCAGTCCTGGCGCCTGACAGGGGGCGCTGACCGCTGCGAGGGGCAGGTGGAGGTA

CACTTCCGAGGGGTCTGGAACACAGTGTGTGACAGTGAGTGGTACCCATCGGAGGCCAAGGTGCTCTGCC

AGTCCTTGGGCTGTGGAACTGCGGTTGAGAGGCCCAAGGGGCTGCCCCACTCCTTGTCCGGCAGGATGTA

CTACTCATGCAATGGGGAGGAGCTCACCCTCTCCAACTGCTCCTGGCGGTTCAACAACTCCAACCTCTGC

AGCCAGTCGCTGGCAGCCAGGGTCCTCTGCTCAGCTTCCCGGAGTTTGCACAATCTGTCCACTCCCGAAG

TCCCTGCAAGTGTTCAGACAGTCACTATAGAATCTTCTGTGACAGTGAAAATAGAGAACAAGGAATCTCG

GGAGCTAATGCTCCTCATCCCCTCCATCGTTCTGGGAATTCTCCTCCTTGGCTCCCTCATCTTCATAGCC

TTCATCCTCTTGAGAATTAAAGGAAAATATGCCCTCCCCGTAATGGTGAACCACCAGCACCTACCCACCA

CCATCCCGGCAGGGAGCAATAGCTATCAACCGGTCCCCATCACCATCCCCAAAGAAGTTTTCATGCTGCC

CATCCAGGTCCAGGCCCCGCCCCCTGAGGACTCAGACTCTGGCTCGGACTCAGACTATGAGCACTATGAC

TTCAGCGCCCAGCCTCCTGTGGCCCTGACCACCTTCTACAATTCCCAGCGGCATCGGGTCACAGATGAGG

AGGTCCAGCAAAGCAGGTTCCAGATGCCACCCTTGGAGGAAGGACTTGAAGAGTTGCATGCCTCCCACAT

CCCAACTGCCAACCCTGGACACTGCATTACAGACCCGCCATCCCTGGGCCCTCAGTATCACCCGAGGAGC

AACAGTGAGTCGAGCACCTCTTCAGGGGAGGATTACTGCAATAGTCCCAAAAGCAAGCTGCCTCCATGGA

ACCCCCAGGTGTTTTCTTCAGAGAGGAGTTCCTTCCTGGAGCAGCCCCCAAACTTGGAGCTGGCCGGCAC

CCAGCCAGCCTTTTCAGCAGGGCCCCCGGCTGATGACAGCTCCAGCACCTCATCCGGGGAGTGGTACCAG

AACTTCCAGCCACCACCCCAGCCCCCTTCGGAGGAGCAGTTTGGCTGTCCAGGGTCCCCCAGCCCTCAGC

CTGACTCCACCGACAACGATGACTACGATGACATCAGCGCAGCCTAG

  

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.

 

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 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 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. J. Immunol. 143:2439 (1989)

2. J. Exp. Med. 174:949 (1991)

3. Eur. J. Immunol. 25:2765 (1995)

4. J. Immunol. 158:1149 (1997)

5. J. Immunol. 178:4351 (2007)

6. Proc. Natl. Acad. Sci. 104:11724 (2007)

 

 

 

Documentation

 

Product datasheet (pdf) can be downloaded here: LTV2579-PDS.pdf