SEMA4D (semaphorin 4D), also known as SEMAJ and CD100, is a single pass type I transmembrane glycoprotein that belongs to the semaphorin family of the immunoglobulin (Ig) superfamily. SEMA4D contains 1 SEMA, 1 PSI, and 1 Ig-like C2-type domain in the extracellular region. SEMA4D exists as a homodimer that utilizes both a membrane proximal intermolecular disulfide bond and two hydrophobic interactions between Phe244 and Phe246.  SEMA4D, produced by T cells, activated B cells and dendritic cells, acts through a low affinity receptor CD72 in the immune system.  SEMA4D enhances B cell survival, macrophages and DC differentiation, and T cell antigen priming.  SEMA4D is a cell surface receptor for PLXN1B and PLXNB2 that plays an important role in cell-cell signaling. SEMA4D promotes reorganization of the actin cytoskeleton and plays a role in axonal growth cone guidance in the developing central nervous system. It regulates dendrite and axon branching and morphogenesis. SEMA4D promotes the migration of cerebellar granule cells and of endothelial cells. SEMA4D also plays a role in the immune system; induces B-cells to aggregate and improves their viability. In the CNS, SEMA4D acts through a high affinity receptor PLXNB1 (Plexin B1) that is expressed in complimentary patterns in several regions of the brain.  Intermediate affinity SEMA4D:PLXNB2 binding also occurs during development, and enhances neuronal migration. SEMA4D can also be produced by tumor associated macrophages, and bind to endothelial cell PLXNB1, thus promoting tumor angiogenesis.

SEMA4D (semaphorin 4D), also known as SEMAJ and CD100, is a single pass type I transmembrane glycoprotein that belongs to the semaphorin family of the immunoglobulin (Ig) superfamily. SEMA4D contains 1 SEMA, 1 PSI, and 1 Ig-like C2-type domain in the extracellular region. SEMA4D exists as a homodimer that utilizes both a membrane proximal intermolecular disulfide bond and two hydrophobic interactions between Phe244 and Phe246.  SEMA4D, produced by T cells, activated B cells and dendritic cells, acts through a low affinity receptor CD72 in the immune system.  SEMA4D enhances B cell survival, macrophages and DC differentiation, and T cell antigen priming.  SEMA4D is a cell surface receptor for PLXN1B and PLXNB2 that plays an important role in cell-cell signaling. SEMA4D promotes reorganization of the actin cytoskeleton and plays a role in axonal growth cone guidance in the developing central nervous system. It regulates dendrite and axon branching and morphogenesis. SEMA4D promotes the migration of cerebellar granule cells and of endothelial cells. SEMA4D also plays a role in the immune system; induces B-cells to aggregate and improves their viability. In the CNS, SEMA4D acts through a high affinity receptor PLXNB1 (Plexin B1) that is expressed in complimentary patterns in several regions of the brain.  Intermediate affinity SEMA4D:PLXNB2 binding also occurs during development, and enhances neuronal migration. SEMA4D can also be produced by tumor associated macrophages, and bind to endothelial cell PLXNB1, thus promoting tumor angiogenesis.

 

Product Details

 

Gene Symbol: SEMA4D; CD100; SEMAJ; coll-4; C9orf164; M-sema-G

 

NCBI Gene ID: 10507

 

Uniprot Entry: Q92854

 

Construct Details: Full length human SEMA4D/CD100 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 +/- a selection marker, see the vector map above)

 

Gene Insert Sequence:  

ATGAGGATGTGCACCCCCATTAGGGGGCTGCTCATGGCCCTTGCAGTGATGTTTGGGACAGCGATGGCAT

TTGCACCCATACCCCGGATCACCTGGGAGCACAGAGAGGTGCACCTGGTGCAGTTTCATGAGCCAGACAT

CTACAACTACTCAGCCTTGCTGCTGAGCGAGGACAAGGACACCTTGTACATAGGTGCCCGGGAGGCGGTC

TTCGCTGTGAACGCACTCAACATCTCCGAGAAGCAGCATGAGGTGTATTGGAAGGTCTCAGAAGACAAAA

AAGCAAAATGTGCAGAAAAGGGGAAATCAAAACAGACAGAGTGCCTCAACTACATCCGGGTGCTGCAGCC

ACTCAGCGCCACTTCCCTTTACGTGTGTGGGACCAACGCATTCCAGCCGGCCTGTGACCACCTGAACTTA

ACATCCTTTAAGTTTCTGGGGAAAAATGAAGATGGCAAAGGAAGATGTCCCTTTGACCCAGCACACAGCT

ACACATCCGTCATGGTTGATGGAGAACTTTATTCGGGGACGTCGTATAATTTTTTGGGAAGTGAACCCAT

CATCTCCCGAAATTCTTCCCACAGTCCTCTGAGGACAGAATATGCAATCCCTTGGCTGAACGAGCCTAGT

TTCGTGTTTGCTGACGTGATCCGAAAAAGCCCAGACAGCCCCGACGGCGAGGATGACAGGGTCTACTTCT

TCTTCACGGAGGTGTCTGTGGAGTATGAGTTTGTGTTCAGGGTGCTGATCCCACGGATAGCAAGAGTGTG

CAAGGGGGACCAGGGCGGCCTGAGGACCTTGCAGAAGAAATGGACCTCCTTCCTGAAAGCCCGACTCATC

TGCTCCCGGCCAGACAGCGGCTTGGTCTTCAATGTGCTGCGGGATGTCTTCGTGCTCAGGTCCCCGGGCC

TGAAGGTGCCTGTGTTCTATGCACTCTTCACCCCACAGCTGAACAACGTGGGGCTGTCGGCAGTGTGCGC

CTACAACCTGTCCACAGCCGAGGAGGTCTTCTCCCACGGGAAGTACATGCAGAGCACCACAGTGGAGCAG

TCCCACACCAAGTGGGTGCGCTATAATGGCCCGGTACCCAAGCCGCGGCCTGGAGCGTGCATCGACAGCG

AGGCACGGGCCGCCAACTACACCAGCTCCTTGAATTTGCCAGACAAGACGCTGCAGTTCGTTAAAGACCA

CCCTTTGATGGATGACTCGGTAACCCCAATAGACAACAGGCCCAGGTTAATCAAGAAAGATGTGAACTAC

ACCCAGATCGTGGTGGACCGGACCCAGGCCCTGGATGGGACTGTCTATGATGTCATGTTTGTCAGCACAG

ACCGGGGAGCTCTGCACAAAGCCATCAGCCTCGAGCACGCTGTTCACATCATCGAGGAGACCCAGCTCTT

CCAGGACTTTGAGCCAGTCCAGACCCTGCTGCTGTCTTCAAAGAAGGGCAACAGGTTTGTCTATGCTGGC

TCTAACTCGGGCGTGGTCCAGGCCCCGCTGGCCTTCTGTGGGAAGCACGGCACCTGCGAGGACTGTGTGC

TGGCGCGGGACCCCTACTGCGCCTGGAGCCCGCCCACAGCGACCTGCGTGGCTCTGCACCAGACCGAGAG

CCCCAGCAGGGGTTTGATTCAGGAGATGAGCGGCGATGCTTCTGTGTGCCCGGATAAAAGTAAAGGAAGT

TACCGGCAGCATTTTTTCAAGCACGGTGGCACAGCGGAACTGAAATGCTCCCAAAAATCCAACCTGGCCC

GGGTCTTTTGGAAGTTCCAGAATGGCGTGTTGAAGGCCGAGAGCCCCAAGTACGGTCTTATGGGCAGAAA

AAACTTGCTCATCTTCAACTTGTCAGAAGGAGACAGTGGGGTGTACCAGTGCCTGTCAGAGGAGAGGGTT

AAGAACAAAACGGTCTTCCAAGTGGTCGCCAAGCACGTCCTGGAAGTGAAGGTGGTTCCAAAGCCCGTAG

TGGCCCCCACCTTGTCAGTTGTTCAGACAGAAGGTAGTAGGATTGCCACCAAAGTGTTGGTGGCATCCAC

CCAAGGGTCTTCTCCCCCAACCCCAGCCGTGCAGGCCACCTCCTCCGGGGCCATCACCCTTCCTCCCAAG

CCTGCGCCCACCGGCACATCCTGCGAACCAAAGATCGTCATCAACACGGTCCCCCAGCTCCACTCGGAGA

AAACCATGTATCTTAAGTCCAGCGACAACCGCCTCCTCATGTCCCTCTTCCTCTTCTTCTTTGTTCTCTT

CCTCTGCCTCTTTTTCTACAACTGCTATAAGGGATACCTGCCCAGACAGTGCTTGAAATTCCGCTCGGCC

CTACTAATTGGGAAGAAGAAGCCCAAGTCAGATTTCTGTGACCGTGAGCAGAGCCTGAAGGAGACGTTAG

TAGAGCCAGGGAGCTTCTCCCAGCAGAATGGGGAGCACCCCAAGCCAGCCCTGGACACCGGCTATGAGAC

CGAGCAAGACACCATCACCAGCAAAGTCCCCACGGATAGGGAGGACTCACAGAGGATCGACGACCTTTCT

GCCAGGGACAAGCCCTTTGACGTCAAGTGTGAGCTGAAGTTCGCTGACTCAGACGCAGATGGAGACTGA

 

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”.

 

 

 

 

 

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

 

References

 

1. Proc. Natl. Acad. Sci. 93:11780-11785(1996)

2. Nature 429:369-374(2004)

3. Cell 99:71-80(1999)

4. Mol. Cell. Biol. 25:6889-6898(2005) 

5. Nature 467:1118-1122(2010)

 

  

 

Product datasheet (pdf) and 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 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.