Human PDGFRB/PDGFR1/CD140b Lentivirus, Full-length Gene in Lentivector, Pre-packaged Lentiviral Particles

Description

PDGFRB (platelet­derived growth factor receptor alpha), also known as CD140b and PDGFR1, is a single-pass type I transmembrane glycoprotein in the CSF-1/PDGF receptor subfamily of the receptor tyrosine kinases (RTK) family. Like other family members, PDGFRB contains 5 immunoglobulin­like (Ig) domains in the extracellular region and a tyrosine kinase domain in the intracellular region. PDGFRB and its closely related homolog PDGFRA can form homo­ or hetero­dimeric receptors when engaged by disulfide­linked dimers of the PDGF family of growth factors, including PDGF-A, B, C or D, or the heterodimer PDGF­AB that is mainly found in human platelets. PDGFRB plays an essential role in the regulation of embryonic development, cell proliferation, survival, differentiation, chemotaxis and migration.  PDGFRB is also required for normal development of the cardiovascular system including blood vessel by promoting proliferation, migration and recruitment of pericytes and smooth muscle cells to endothelial cells. PDGFRB is important for normal formation of a branched network of capillaries in kidney glomeruli. PDGFRB promotes rearrangement of the actin cytoskeleton and the formation of membrane ruffles. Binding of its cognate ligands, such ashomodimeric PDGFB, heterodimers formed by PDGFA and PDGFB or homodimeric PDGFD, leads to the activation of several signaling cascades.  A translocation between chromosomes 5 and 12, that fuses the PDGFRB gene to that of the translocation, ETV6, leukemia gene, results in chronic myeloproliferative disorder with eosinophilia.

 

 

Gene Symbol: CD140B; PDGFRB; PDGFR1; PDGF-R-beta; IMF1; KOGS; IBGC4; JTK12; PDGFR; PENTT; PDGFR-1

 

NCBI Gene ID: 5159

 

Uniprot Entry: P09619

 

Construct Details: Full length human PDGFRB/CD140b gene is subcloned into the Lentiviral expression vector pLTC with an upstream CMV promoter 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.

 

Vector Type: pLTC or pLTC-IRES-Marker (lentiviral expression vector containing a heterologous CMV promoter with/without a selection marker, see the vector map above)

 

Gene Insert Sequence:  

ATGCGGCTTCCGGGTGCGATGCCAGCTCTGGCCCTCAAAGGCGAGCTGCTGTTGCTGTCTCTCCTGTTAC

TTCTGGAACCACAGATCTCTCAGGGCCTGGTCGTCACACCCCCGGGGCCAGAGCTTGTCCTCAATGTCTC

CAGCACCTTCGTTCTGACCTGCTCGGGTTCAGCTCCGGTGGTGTGGGAACGGATGTCCCAGGAGCCCCCA

CAGGAAATGGCCAAGGCCCAGGATGGCACCTTCTCCAGCGTGCTCACACTGACCAACCTCACTGGGCTAG

ACACGGGAGAATACTTTTGCACCCACAATGACTCCCGTGGACTGGAGACCGATGAGCGGAAACGGCTCTA

CATCTTTGTGCCAGATCCCACCGTGGGCTTCCTCCCTAATGATGCCGAGGAACTATTCATCTTTCTCACG

GAAATAACTGAGATCACCATTCCATGCCGAGTAACAGACCCACAGCTGGTGGTGACACTGCACGAGAAGA

AAGGGGACGTTGCACTGCCTGTCCCCTATGATCACCAACGTGGCTTTTCTGGTATCTTTGAGGACAGAAG

CTACATCTGCAAAACCACCATTGGGGACAGGGAGGTGGATTCTGATGCCTACTATGTCTACAGACTCCAG

GTGTCATCCATCAACGTCTCTGTGAACGCAGTGCAGACTGTGGTCCGCCAGGGTGAGAACATCACCCTCA

TGTGCATTGTGATCGGGAATGAGGTGGTCAACTTCGAGTGGACATACCCCCGCAAAGAAAGTGGGCGGCT

GGTGGAGCCGGTGACTGACTTCCTCTTGGATATGCCTTACCACATCCGCTCCATCCTGCACATCCCCAGT

GCCGAGTTAGAAGACTCGGGGACCTACACCTGCAATGTGACGGAGAGTGTGAATGACCATCAGGATGAAA

AGGCCATCAACATCACCGTGGTTGAGAGCGGCTACGTGCGGCTCCTGGGAGAGGTGGGCACACTACAATT

TGCTGAGCTGCATCGGAGCCGGACACTGCAGGTAGTGTTCGAGGCCTACCCACCGCCCACTGTCCTGTGG

TTCAAAGACAACCGCACCCTGGGCGACTCCAGCGCTGGCGAAATCGCCCTGTCCACGCGCAACGTGTCGG

AGACCCGGTATGTGTCAGAGCTGACACTGGTTCGCGTGAAGGTGGCAGAGGCTGGCCACTACACCATGCG

GGCCTTCCATGAGGATGCTGAGGTCCAGCTCTCCTTCCAGCTACAGATCAATGTCCCTGTCCGAGTGCTG

GAGCTAAGTGAGAGCCACCCTGACAGTGGGGAACAGACAGTCCGCTGTCGTGGCCGGGGCATGCCCCAGC

CGAACATCATCTGGTCTGCCTGCAGAGACCTCAAAAGGTGTCCACGTGAGCTGCCGCCCACGCTGCTGGG

GAACAGTTCCGAAGAGGAGAGCCAGCTGGAGACTAACGTGACGTACTGGGAGGAGGAGCAGGAGTTTGAG

GTGGTGAGCACACTGCGTCTGCAGCACGTGGATCGGCCACTGTCGGTGCGCTGCACGCTGCGCAACGCTG

TGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAAGGTGGTGGTGATCTCAGC

CATCCTGGCCCTGGTGGTGCTCACCATCATCTCCCTTATCATCCTCATCATGCTTTGGCAGAAGAAGCCA

CGTTACGAGATCCGATGGAAGGTGATTGAGTCTGTGAGCTCTGACGGCCATGAGTACATCTACGTGGACC

CCATGCAGCTGCCCTATGACTCCACGTGGGAGCTGCCGCGGGACCAGCTTGTGCTGGGACGCACCCTCGG

CTCTGGGGCCTTTGGGCAGGTGGTGGAGGCCACGGCTCATGGCCTGAGCCATTCTCAGGCCACGATGAAA

GTGGCCGTCAAGATGCTTAAATCCACAGCCCGCAGCAGTGAGAAGCAAGCCCTTATGTCGGAGCTGAAGA

TCATGAGTCACCTTGGGCCCCACCTGAACGTGGTCAACCTGTTGGGGGCCTGCACCAAAGGAGGACCCAT

CTATATCATCACTGAGTACTGCCGCTACGGAGACCTGGTGGACTACCTGCACCGCAACAAACACACCTTC

CTGCAGCACCACTCCGACAAGCGCCGCCCGCCCAGCGCGGAGCTCTACAGCAATGCTCTGCCCGTTGGGC

TCCCCCTGCCCAGCCATGTGTCCTTGACCGGGGAGAGCGACGGTGGCTACATGGACATGAGCAAGGACGA

GTCGGTGGACTATGTGCCCATGCTGGACATGAAAGGAGACGTCAAATATGCAGACATCGAGTCCTCCAAC

TACATGGCCCCTTACGATAACTACGTTCCCTCTGCCCCTGAGAGGACCTGCCGAGCAACTTTGATCAACG

AGTCTCCAGTGCTAAGCTACATGGACCTCGTGGGCTTCAGCTACCAGGTGGCCAATGGCATGGAGTTTCT

GGCCTCCAAGAACTGCGTCCACAGAGACCTGGCGGCTAGGAACGTGCTCATCTGTGAAGGCAAGCTGGTC

AAGATCTGTGACTTTGGCCTGGCTCGAGACATCATGCGGGACTCGAATTACATCTCCAAAGGCAGCACCT

TTTTGCCTTTAAAGTGGATGGCTCCGGAGAGCATCTTCAACAGCCTCTACACCACCCTGAGCGACGTGTG

GTCCTTCGGGATCCTGCTCTGGGAGATCTTCACCTTGGGTGGCACCCCTTACCCAGAGCTGCCCATGAAC

GAGCAGTTCTACAATGCCATCAAACGGGGTTACCGCATGGCCCAGCCTGCCCATGCCTCCGACGAGATCT

ATGAGATCATGCAGAAGTGCTGGGAAGAGAAGTTTGAGATTCGGCCCCCCTTCTCCCAGCTGGTGCTGCT

TCTCGAGAGACTGTTGGGCGAAGGTTACAAAAAGAAGTACCAGCAGGTGGATGAGGAGTTTCTGAGGAGT

GACCACCCAGCCATCCTTCGGTCCCAGGCCCGCTTGCCTGGGTTCCATGGCCTCCGATCTCCCCTGGACA

CCAGCTCCGTCCTCTATACTGCCGTGCAGCCCAATGAGGGTGACAACGACTATATCATCCCCCTGCCTGA

CCCCAAACCCGAGGTTGCTGACGAGGGCCCACTGGAGGGTTCCCCCAGCCTAGCCAGCTCCACCCTGAAT

GAAGTCAACACCTCCTCAACCATCTCCTGTGACAGCCCCCTGGAGCCCCAGGACGAACCAGAGCCAGAGC

CCCAGCTTGAGCTCCAGGTGGAGCCGGAGCCAGAGCTGGAACAGTTGCCGGATTCGGGGTGCCCTGCGCC

TCGGGCGGAAGCAGAGGATAGCTTCCTGTAG

 

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