Human PTPRC/CD45/LCA/LY5/B220/T200 Lentivirus, Full-length Gene in Lentivector, Pre-packaged lentiviral Particles

Description

PTPRC (protein tyrosine phosphatase, receptor type C) or CD45 is a single-pass type I transmembrane protein belonging to the receptor class 1/6 subfamily of the protein-tyrosine phosphatase (PTP) family.  PTPs regulate a variety of cellular processes including cell growth, differentiation, mitosis, and oncogenic transformation. PTPRC/CD45 contains an extracellular domain (ECD) with 2 fibronectin type-III motifs, a single transmembrane (TM) segment and two tandem intracytoplasmic catalytic domains (ICD), and thus is classified as a receptor type PTP.  The first PTPase domain has enzymatic activity, while the second one seems to affect the substrate specificity of the first one.  PTPRC/CD45 is an essential regulator of T- and B-cell antigen receptor signaling. PTPRC/CD45 functions through either direct interaction with components of the antigen receptor complexes, or by activating various Src family kinases required for the antigen receptor signaling. PTPRC/CD45 also suppresses JAK kinases, and thus functions as a regulator of cytokine receptor signaling. Diseases associated with PTPRC/CD45 include Severe Combined Immunodeficiency, T Cell-Negative, B-Cell/Natural Killer-Cell Positive and Hepatitis C Virus. 

 

 

Gene Symbol: PTPRC; LCA; LY5; B220; CD45; L-CA; T200; CD45R; GP180

 

NCBI Gene ID: 5788

 

Uniprot Entry: P08575

 

Construct Details: Full length human PTPRC/CD45 gene is subcloned into the Lentiviral expression vector pLTC with an upstream CMV promoter and with or without an antibiotic 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: 3915 (bp)

 

Gene Insert Sequence:  

ATGTATTTGTGGCTTAAACTCTTGGCATTTGGCTTTGCCTTTCTGGACACAGAAGTATTTGTGACAGGGCAAAGCCCAAC

ACCTTCCCCCACTGGATTGACTACAGCAAAGATGCCCAGTGTTCCACTTTCAAGTGACCCCTTACCTACTCACACCACTG

CATTCTCACCCGCAAGCACCTTTGAAAGAGAAAATGACTTCTCAGAGACCACAACTTCTCTTAGTCCAGACAATACTTCC

ACCCAAGTATCCCCGGACTCTTTGGATAATGCTAGTGCTTTTAATACCACAGGTGTTTCATCAGTACAGACGCCTCACCT

TCCCACGCACGCAGACTCGCAGACGCCCTCTGCTGGAACTGACACGCAGACATTCAGCGGCTCCGCCGCCAATGCAAAAC

TCAACCCTACCCCAGGCAGCAATGCTATCTCAGATGTCCCAGGAGAGAGGAGTACAGCCAGCACCTTTCCTACAGACCCA

GTTTCCCCATTGACAACCACCCTCAGCCTTGCACACCACAGCTCTGCTGCCTTACCTGCACGCACCTCCAACACCACCAT

CACAGCGAACACCTCAGATGCCTACCTTAATGCCTCTGAAACAACCACTCTGAGCCCTTCTGGAAGCGCTGTCATTTCAA

CCACAACAATAGCTACTACTCCATCTAAGCCAACATGTGATGAAAAATATGCAAACATCACTGTGGATTACTTATATAAC

AAGGAAACTAAATTATTTACAGCAAAGCTAAATGTTAATGAGAATGTGGAATGTGGAAACAATACTTGCACAAACAATGA

GGTGCATAACCTTACAGAATGTAAAAATGCGTCTGTTTCCATATCTCATAATTCATGTACTGCTCCTGATAAGACATTAA

TATTAGATGTGCCACCAGGGGTTGAAAAGTTTCAGTTACATGATTGTACACAAGTTGAAAAAGCAGATACTACTATTTGT

TTAAAATGGAAAAATATTGAAACCTTTACTTGTGATACACAGAATATTACCTACAGATTTCAGTGTGGTAATATGATATT

TGATAATAAAGAAATTAAATTAGAAAACCTTGAACCCGAACATGAGTATAAGTGTGACTCAGAAATACTCTATAATAACC

ACAAGTTTACTAACGCAAGTAAAATTATTAAAACAGATTTTGGGAGTCCAGGAGAGCCTCAGATTATTTTTTGTAGAAGT

GAAGCTGCACATCAAGGAGTAATTACCTGGAATCCCCCTCAAAGATCATTTCATAATTTTACCCTCTGTTATATAAAAGA

GACAGAAAAAGATTGCCTCAATCTGGATAAAAACCTGATCAAATATGATTTGCAAAATTTAAAACCTTATACGAAATATG

TTTTATCATTACATGCCTACATCATTGCAAAAGTGCAACGTAATGGAAGTGCTGCAATGTGTCATTTCACAACTAAAAGT

GCTCCTCCAAGCCAGGTCTGGAACATGACTGTCTCCATGACATCAGATAATAGTATGCATGTCAAGTGTAGGCCTCCCAG

GGACCGTAATGGCCCCCATGAACGTTACCATTTGGAAGTTGAAGCTGGAAATACTCTGGTTAGAAATGAGTCGCATAAGA

ATTGCGATTTCCGTGTAAAAGATCTTCAATATTCAACAGACTACACTTTTAAGGCCTATTTTCACAATGGAGACTATCCT

GGAGAACCCTTTATTTTACATCATTCAACATCTTATAATTCTAAGGCACTGATAGCATTTCTGGCATTTCTGATTATTGT

GACATCAATAGCCCTGCTTGTTGTTCTCTACAAAATCTATGATCTACATAAGAAAAGATCCTGCAATTTAGATGAACAGC

AGGAGCTTGTTGAAAGGGATGATGAAAAACAACTGATGAATGTGGAGCCAATCCATGCAGATATTTTGTTGGAAACTTAT

AAGAGGAAGATTGCTGATGAAGGAAGACTTTTTCTGGCTGAATTTCAGAGCATCCCGCGGGTGTTCAGCAAGTTTCCTAT

AAAGGAAGCTCGAAAGCCCTTTAACCAGAATAAAAACCGTTATGTTGACATTCTTCCTTATGATTATAACCGTGTTGAAC

TCTCTGAGATAAACGGAGATGCAGGGTCAAACTACATAAATGCCAGCTATATTGATGGTTTCAAAGAACCCAGGAAATAC

ATTGCTGCACAAGGTCCCAGGGATGAAACTGTTGATGATTTCTGGAGGATGATTTGGGAACAGAAAGCCACAGTTATTGT

CATGGTCACTCGATGTGAAGAAGGAAACAGGAACAAGTGTGCAGAATACTGGCCGTCAATGGAAGAGGGCACTCGGGCTT

TTGGAGATGTTGTTGTAAAGATCAACCAGCACAAAAGATGTCCAGATTACATCATTCAGAAATTGAACATTGTAAATAAA

AAAGAAAAAGCAACTGGAAGAGAGGTGACTCACATTCAGTTCACCAGCTGGCCAGACCACGGGGTGCCTGAGGATCCTCA

CTTGCTCCTCAAACTGAGAAGGAGAGTGAATGCCTTCAGCAATTTCTTCAGTGGTCCCATTGTGGTGCACTGCAGTGCTG

GTGTTGGGCGCACAGGAACCTATATCGGAATTGATGCCATGCTAGAAGGCCTGGAAGCCGAGAACAAAGTGGATGTTTAT

GGTTATGTTGTCAAGCTAAGGCGACAGAGATGCCTGATGGTTCAAGTAGAGGCCCAGTACATCTTGATCCATCAGGCTTT

GGTGGAATACAATCAGTTTGGAGAAACAGAAGTGAATTTGTCTGAATTACATCCATATCTACATAACATGAAGAAAAGGG

ATCCACCCAGTGAGCCGTCTCCACTAGAGGCTGAATTCCAGAGACTTCCTTCATATAGGAGCTGGAGGACACAGCACATT

GGAAATCAAGAAGAAAATAAAAGTAAAAACAGGAATTCTAATGTCATCCCATATGACTATAACAGAGTGCCACTTAAACA

TGAGCTGGAAATGAGTAAAGAGAGTGAGCATGATTCAGATGAATCCTCTGATGATGACAGTGATTCAGAGGAACCAAGCA

AATACATCAATGCATCTTTTATAATGAGCTACTGGAAACCTGAAGTGATGATTGCTGCTCAGGGACCACTGAAGGAGACC

ATTGGTGACTTTTGGCAGATGATCTTCCAAAGAAAAGTCAAAGTTATTGTTATGCTGACAGAACTGAAACATGGAGACCA

GGAAATCTGTGCTCAGTACTGGGGAGAAGGAAAGCAAACATATGGAGATATTGAAGTTGACCTGAAAGACACAGACAAAT

CTTCAACTTATACCCTTCGTGTCTTTGAACTGAGACATTCCAAGAGGAAAGACTCTCGAACTGTGTACCAGTACCAATAT

ACAAACTGGAGTGTGGAGCAGCTTCCTGCAGAACCCAAGGAATTAATCTCTATGATTCAGGTCGTCAAACAAAAACTTCC

CCAGAAGAATTCCTCTGAAGGGAACAAGCATCACAAGAGTACACCTCTACTCATTCACTGCAGGGATGGATCTCAGCAAA

CGGGAATATTTTGTGCTTTGTTAAATCTCTTAGAAAGTGCGGAAACAGAAGAGGTAGTGGATATTTTTCAAGTGGTAAAA

GCTCTACGCAAAGCTAGGCCAGGCATGGTTTCCACATTCGAGCAATATCAATTCCTATATGACGTCATTGCCAGCACCTA

CCCTGCTCAGAATGGACAAGTAAAGAAAAACAACCATCAAGAAGATAAAATTGAATTTGATAATGAAGTGGACAAAGTAA

AGCAGGATGCTAATTGTGTTAATCCACTTGGTGCCCCAGAAAAGCTCCCTGAAGCAAAGGAACAGGCTGAAGGTTCTGAA

CCCACGAGTGGCACTGAGGGGCCAGAACATTCTGTCAATGGTCCTGCAAGTCCAGCTTTAAATCAAGGTTCATAG

 

Formulation: pre-packaged viral 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”.

 

 

 

 

 

 

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 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

 

 

 

 

 

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