Vascular endothelial cell growth factors (VEGFs) are secreted polypeptides with a highly conserved cystine-knot structure similar to that of the platelet-derived growth factors (PDGFs).  VEGFs play important roles in vascular development and in diseases involving abnormal growth of blood vessels such as tumor-related angiogenesis. VEGFs stimulate angiogenesis by binding to the specific receptors (VEGFRs) on the surface of vascular endothelial cells. All the three VEGFRs, VEGFR1 (Flt-1), VEGFR2 (KDR/Flk-1/CD309), and VEGFR3 (Flt-4), belong to the CSF-1/PDGF receptor subfamily of receptor tyrosine kinase superfamily (RTKs). Each VEGFR contains 7 immunoglobulin (Ig)-like C2-type domains in the extracellular region and an intracellular kinase domain. VEGFR3 is widely expressed in the early embryo but restricted to lymphatic vessels at later stages of development. VEGFR3 is up-regulated in tumor angiogenesis. VEGFC and VEGFD have been identified as ligands for VEGFR3. VEGFR3 plays a significant role in ligand-mediated lymphangiogenesis. Mutation in this gene is associated with the primary lymphedema, a rare genetic condition with both autosomal dominant and autosomal recessive modes of inheritance. The VEGFR3-mediated signaling pathway may provide a target for anti-lymphangiogenic therapy in cancer.

Vascular endothelial cell growth factors (VEGFs) are secreted polypeptides with a highly conserved cystine-knot structure similar to that of the platelet-derived growth factors (PDGFs).  VEGFs play important roles in vascular development and in diseases involving abnormal growth of blood vessels such as tumor-related angiogenesis. VEGFs stimulate angiogenesis by binding to the specific receptors (VEGFRs) on the surface of vascular endothelial cells. All the three VEGFRs, VEGFR1 (Flt-1), VEGFR2 (KDR/Flk-1/CD309), and VEGFR3 (Flt-4), belong to the CSF-1/PDGF receptor subfamily of receptor tyrosine kinase superfamily (RTKs). Each VEGFR contains 7 immunoglobulin (Ig)-like C2-type domains in the extracellular region and an intracellular kinase domain. VEGFR3 is widely expressed in the early embryo but restricted to lymphatic vessels at later stages of development. VEGFR3 is up-regulated in tumor angiogenesis. VEGFC and VEGFD have been identified as ligands for VEGFR3. VEGFR3 plays a significant role in ligand-mediated lymphangiogenesis. Mutation in this gene is associated with the primary lymphedema, a rare genetic condition with both autosomal dominant and autosomal recessive modes of inheritance. The VEGFR3-mediated signaling pathway may provide a target for anti-lymphangiogenic therapy in cancer.

 

Product Details

 

Gene Symbol: FLT4; PCL; FLT-4; LMPH1A; VEGFR3

 

NCBI Gene ID: 2324

 

Uniprot Entry: P35916

 

Construct Details: Full length human FLT4 gene (encoding Uniprot#P35916-1) 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 Sequence:  

ATGCAGCGGGGCGCCGCGCTGTGCCTGCGACTGTGGCTCTGCCTGGGACTCCTGGACGGCCTGGTGAGTG

GCTACTCCATGACCCCCCCGACCTTGAACATCACGGAGGAGTCACACGTCATCGACACCGGTGACAGCCT

GTCCATCTCCTGCAGGGGACAGCACCCCCTCGAGTGGGCTTGGCCAGGAGCTCAGGAGGCGCCAGCCACC

GGAGACAAGGACAGCGAGGACACGGGGGTGGTGCGAGACTGCGAGGGCACAGACGCCAGGCCCTACTGCA

AGGTGTTGCTGCTGCACGAGGTACATGCCAACGACACAGGCAGCTACGTCTGCTACTACAAGTACATCAA

GGCACGCATCGAGGGCACCACGGCCGCCAGCTCCTACGTGTTCGTGAGAGACTTTGAGCAGCCATTCATC

AACAAGCCTGACACGCTCTTGGTCAACAGGAAGGACGCCATGTGGGTGCCCTGTCTGGTGTCCATCCCCG

GCCTCAATGTCACGCTGCGCTCGCAAAGCTCGGTGCTGTGGCCAGACGGGCAGGAGGTGGTGTGGGATGA

CCGGCGGGGCATGCTCGTGTCCACGCCACTGCTGCACGATGCCCTGTACCTGCAGTGCGAGACCACCTGG

GGAGACCAGGACTTCCTTTCCAACCCCTTCCTGGTGCACATCACAGGCAACGAGCTCTATGACATCCAGC

TGTTGCCCAGGAAGTCGCTGGAGCTGCTGGTAGGGGAGAAGCTGGTCCTGAACTGCACCGTGTGGGCTGA

GTTTAACTCAGGTGTCACCTTTGACTGGGACTACCCAGGGAAGCAGGCAGAGCGGGGTAAGTGGGTGCCC

GAGCGACGCTCCCAGCAGACCCACACAGAACTCTCCAGCATCCTGACCATCCACAACGTCAGCCAGCACG

ACCTGGGCTCGTATGTGTGCAAGGCCAACAACGGCATCCAGCGATTTCGGGAGAGCACCGAGGTCATTGT

GCATGAAAATCCCTTCATCAGCGTCGAGTGGCTCAAAGGACCCATCCTGGAGGCCACGGCAGGAGACGAG

CTGGTGAAGCTGCCCGTGAAGCTGGCAGCGTACCCCCCGCCCGAGTTCCAGTGGTACAAGGATGGAAAGG

CACTGTCCGGGCGCCACAGTCCACATGCCCTGGTGCTCAAGGAGGTGACAGAGGCCAGCACAGGCACCTA

CACCCTCGCCCTGTGGAACTCCGCTGCTGGCCTGAGGCGCAACATCAGCCTGGAGCTGGTGGTGAATGTG

CCCCCCCAGATACATGAGAAGGAGGCCTCCTCCCCCAGCATCTACTCGCGTCACAGCCGCCAGGCCCTCA

CCTGCACGGCCTACGGGGTGCCCCTGCCTCTCAGCATCCAGTGGCACTGGCGGCCCTGGACACCCTGCAA

GATGTTTGCCCAGCGTAGTCTCCGGCGGCGGCAGCAGCAAGACCTCATGCCACAGTGCCGTGACTGGAGG

GCGGTGACCACGCAGGATGCCGTGAACCCCATCGAGAGCCTGGACACCTGGACCGAGTTTGTGGAGGGAA

AGAATAAGACTGTGAGCAAGCTGGTGATCCAGAATGCCAACGTGTCTGCCATGTACAAGTGTGTGGTCTC

CAACAAGGTGGGCCAGGATGAGCGGCTCATCTACTTCTATGTGACCACCATCCCCGACGGCTTCACCATC

GAATCCAAGCCATCCGAGGAGCTACTAGAGGGCCAGCCGGTGCTCCTGAGCTGCCAAGCCGACAGCTACA

AGTACGAGCATCTGCGCTGGTACCGCCTCAACCTGTCCACGCTGCACGATGCGCACGGGAACCCGCTTCT

GCTCGACTGCAAGAACGTGCATCTGTTCGCCACCCCTCTGGCCGCCAGCCTGGAGGAGGTGGCACCTGGG

GCGCGCCACGCCACGCTCAGCCTGAGTATCCCCCGCGTCGCGCCCGAGCACGAGGGCCACTATGTGTGCG

AAGTGCAAGACCGGCGCAGCCATGACAAGCACTGCCACAAGAAGTACCTGTCGGTGCAGGCCCTGGAAGC

CCCTCGGCTCACGCAGAACTTGACCGACCTCCTGGTGAACGTGAGCGACTCGCTGGAGATGCAGTGCTTG

GTGGCCGGAGCGCACGCGCCCAGCATCGTGTGGTACAAAGACGAGAGGCTGCTGGAGGAAAAGTCTGGAG

TCGACTTGGCGGACTCCAACCAGAAGCTGAGCATCCAGCGCGTGCGCGAGGAGGATGCGGGACGCTATCT

GTGCAGCGTGTGCAACGCCAAGGGCTGCGTCAACTCCTCCGCCAGCGTGGCCGTGGAAGGCTCCGAGGAT

AAGGGCAGCATGGAGATCGTGATCCTTGTCGGTACCGGCGTCATCGCTGTCTTCTTCTGGGTCCTCCTCC

TCCTCATCTTCTGTAACATGAGGAGGCCGGCCCACGCAGACATCAAGACGGGCTACCTGTCCATCATCAT

GGACCCCGGGGAGGTGCCTCTGGAGGAGCAATGCGAATACCTGTCCTACGATGCCAGCCAGTGGGAATTC

CCCCGAGAGCGGCTGCACCTGGGGAGAGTGCTCGGCTACGGCGCCTTCGGGAAGGTGGTGGAAGCCTCCG

CTTTCGGCATCCACAAGGGCAGCAGCTGTGACACCGTGGCCGTGAAAATGCTGAAAGAGGGCGCCACGGC

CAGCGAGCACCGCGCGCTGATGTCGGAGCTCAAGATCCTCATTCACATCGGCAACCACCTCAACGTGGTC

AACCTCCTCGGGGCGTGCACCAAGCCGCAGGGCCCCCTCATGGTGATCGTGGAGTTCTGCAAGTACGGCA

ACCTCTCCAACTTCCTGCGCGCCAAGCGGGACGCCTTCAGCCCCTGCGCGGAGAAGTCTCCCGAGCAGCG

CGGACGCTTCCGCGCCATGGTGGAGCTCGCCAGGCTGGATCGGAGGCGGCCGGGGAGCAGCGACAGGGTC

CTCTTCGCGCGGTTCTCGAAGACCGAGGGCGGAGCGAGGCGGGCTTCTCCAGACCAAGAAGCTGAGGACC

TGTGGCTGAGCCCGCTGACCATGGAAGATCTTGTCTGCTACAGCTTCCAGGTGGCCAGAGGGATGGAGTT

CCTGGCTTCCCGAAAGTGCATCCACAGAGACCTGGCTGCTCGGAACATTCTGCTGTCGGAAAGCGACGTG

GTGAAGATCTGTGACTTTGGCCTTGCCCGGGACATCTACAAAGACCCCGACTACGTCCGCAAGGGCAGTG

CCCGGCTGCCCCTGAAGTGGATGGCCCCTGAAAGCATCTTCGACAAGGTGTACACCACGCAGAGTGACGT

GTGGTCCTTTGGGGTGCTTCTCTGGGAGATCTTCTCTCTGGGGGCCTCCCCGTACCCTGGGGTGCAGATC

AATGAGGAGTTCTGCCAGCGGCTGAGAGACGGCACAAGGATGAGGGCCCCGGAGCTGGCCACTCCCGCCA

TACGCCGCATCATGCTGAACTGCTGGTCCGGAGACCCCAAGGCGAGACCTGCATTCTCGGAGCTGGTGGA

GATCCTGGGGGACCTGCTCCAGGGCAGGGGCCTGCAAGAGGAAGAGGAGGTCTGCATGGCCCCGCGCAGC

TCTCAGAGCTCAGAAGAGGGCAGCTTCTCGCAGGTGTCCACCATGGCCCTACACATCGCCCAGGCTGACG

CTGAGGACAGCCCGCCAAGCCTGCAGCGCCACAGCCTGGCCGCCAGGTATTACAACTGGGTGTCCTTTCC

CGGGTGCCTGGCCAGAGGGGCTGAGACCCGTGGTTCCTCCAGGATGAAGACATTTGAGGAATTCCCCATG

ACCCCAACGACCTACAAAGGCTCTGTGGACAACCAGACAGACAGTGGGATGGTGCTGGCCTCGGAGGAGT

TTGAGCAGATAGAGAGCAGGCATAGACAAGAAAGCGGCTTCAGGTAG

 

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