Human IgSF3/EWI3/LCDD Lentivirus, Full-length Gene in Lentivector, Pre-packaged lentiviral Particles

Description

IgSF2 or EWI3 is a 1194 amino acid (aa) single pass type I transmembrane glycoprotein that belongs to the EWI subfamily of the Immunoglobulin superfamily (IgSF).  IgSF3 contains an N-terminal signal peptide, a 1105-aa extracellular domain (ECD) with 8 Ig-like C2-type domains, an EWI (Glu-Trp-Ile)-motif and 5 potential glycosylation sites, a transmembrane domain (TM), and a 49-aa cytoplasmic tail. IThere is likely an interchromosomal Alu-mediated fusion between IGSF3 on 1p13.1 and GGT on 22q11.2.  A mutation in the IgSF3 gene has been associated with bilateral nasolacrimal duct obstruction (LCDD), a condition resulting in the imbalance between tear production and tear drainage caused by failure of the nasolacrimal duct to open into the inferior meatus.

 

 

Gene Symbol: IgSF3; EWI3; V8; LCDD; EWI-3; IGSF-3

 

NCBI Gene ID: 3321

 

Uniprot Entry: O75054

 

Construct Details: Full length human IgSF3 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 Size: 3585 (bp)

 

Gene Insert Sequence:  

ATGAAGTGCTTTTTCCCGGTGCTGAGCTGTCTGGCTGTGCTGGGTGTGGTGTCAGCACAGCGGCAGGTCA

CCGTTCAGGAAGGACCCTTGTACCGCACGGAGGGCTCCCACATCACTATCTGGTGCAATGTGAGTGGCTA

CCAGGGACCTTCTGAGCAGAATTTCCAGTGGTCCATTTACCTGCCTTCGTCGCCAGAGCGAGAGGTGCAG

ATCGTCAGCACCATGGACTCTTCCTTCCCCTATGCCATCTACACCCAGCGCGTCCGCGGAGGGAAGATCT

TCATAGAAAGAGTCCAGGGGAACTCAACCCTATTGCACATCACAGATCTTCAGGCCCGGGATGCCGGGGA

GTATGAATGCCACACACCCAGCACTGATAAGCAATACTTTGGGAGTTACAGTGCAAAGATGAACCTAGTG

GTGATCCCAGACTCCCTGCAGACCACTGCCATGCCCCAGACTCTGCACAGAGTGGAGCAGGACCCGCTGG

AGCTCACTTGTGAGGTGGCCTCAGAGACCATTCAGCACAGCCACCTGTCTGTGGCCTGGCTCCGGCAGAA

AGTTGGCGAGAAGCCCGTGGAGGTCATCTCCCTGAGCCGAGATTTCATGCTTCACTCCAGCAGCGAATAT

GCCCAGAGGCAGAGCCTGGGGGAGGTGCGGCTGGACAAGCTGGGGAGGACCACCTTCCGCCTCACCATCT

TCCACCTGCAGCCTTCTGACCAGGGCGAATTCTACTGCGAGGCCGCCGAGTGGATCCAGGATCCGGATGG

GTCGTGGTATGCTATGACCCGAAAGCGTTCCGAGGGAGCCGTGGTCAACGTCCAGCCAACTGACAAAGAA

TTCACTGTTCGGCTGGAGACAGAGAAGCGGCTGCACACGGTGGGCGAGCCGGTGGAGTTCAGATGCATCC

TGGAGGCTCAGAATGTTCCCGACCGTTACTTTGCTGTCTCCTGGGCCTTCAACAGCTCGCTCATCGCCAC

CATGGGTCCTAACGCTGTGCCTGTCCTCAACAGCGAATTTGCTCACCGGGAAGCCAGGGGACAGCTTAAG

GTGGCCAAAGAGAGCGACAGTGTCTTTGTGCTGAAGATCTACCACCTCCGCCAGGAAGATAGCGGGAAAT

ACAACTGCCGGGTGACTGAGCGAGAGAAAACCGTGACCGGGGAATTCATTGATAAGGAGAGCAAGCGTCC

CAAGAACATCCCCATCATAGTCCTCCCCCTCAAGAGCAGCATCTCCGTGGAGGTGGCCAGCAATGCCAGC

GTCATCCTTGAGGGCGAGGACCTGCGCTTCTCCTGCAGTGTCCGCACGGCAGGCAGGCCGCAGGGTCGCT

TCTCTGTCATCTGGCAGCTTGTGGACAGGCAGAACCGCCGCAGCAATATCATGTGGCTAGACCGGGATGG

CACCGTGCAGCCAGGCTCGTCCTACTGGGAGCGCAGCAGCTTTGGGGGCGTCCAGATGGAGCAGGTGCAG

CCCAACTCGTTCAGCCTGGGCATCTTCAACAGCAGGAAGGAGGACGAGGGCCAGTATGAATGCCATGTGA

CTGAATGGGTGCGGGCAGTGGATGGCGAGTGGCAGATTGTTGGGGAGCGCCGGGCCAGCACTCCCATCTC

CATCACAGCTCTTGAAATGGGCTTCGCAGTCACAGCCATCTCCCGGACACCGGGGGTGACCTACAGCGAC

TCCTTTGACTTGCAGTGTATCATCAAACCCCACTACCCTGCCTGGGTCCCCGTGTCGGTGACATGGCGGT

TCCAGCCGGTGGGCACGGTGGAGTTCCATGACTTGGTGACCTTCACCCGGGACGGAGGGGTCCAGTGGGG

GGACAGGTCCTCCAGCTTCCGAACCCGAACTGCCATCGAGAAGGCTGAGTCCAGCAACAACGTCCGCCTA

AGCATCAGCCGAGCCAGTGACACGGAAGCAGGCAAGTACCAGTGTGTGGCAGAGCTGTGGCGGAAGAACT

ACAACAACACCTGGACGCGACTGGCGGAGAGGACCTCCAACCTGCTGGAGATCAGGGTGCTGCAGCCAGT

GACAAAGCTGCAGGTGAGCAAATCGAAGAGGACCCTCACCCTGGTGGAAAACAAGCCCATTCAGTTGAAC

TGCTCAGTCAAGTCTCAGACTAGCCAGAACTCCCACTTTGCGGTGCTCTGGTATGTCCACAAGCCCTCGG

ATGCCGATGGCAAGCTTATCCTGAAGACCACCCACAACTCCGCCTTTGAATACGGTACTTACGCCGAGGA

GGAGGGCCTGAGAGCCAGGCTCCAGTTTGAGAGGCATGTGTCGGGGGGCCTGTTCAGCCTCACCGTCCAG

AGAGCCGAGGTCAGCGACAGCGGCAGCTACTACTGCCACGTGGAGGAGTGGCTGCTGAGCCCCAACTACG

CCTGGTACAAGCTGGCAGAGGAGGTTTCTGGGCGCACAGAAGTCACTGTGAAACAGCCAGACAGCCGCCT

GAGGCTCAGCCAAGCCCAGGGGAACCTGTCGGTTCTGGAGACCCGGCAGGTACAGCTGGAGTGTGTGGTT

CTCAACCGCACCAGCATAACCTCCCAGCTCATGGTGGAATGGTTTGTATGGAAGCCCAACCACCCTGAGC

GGGAGACTGTGGCCCGCTTGAGCCGTGACGCCACCTTCCACTATGGAGAGCAGGCAGCCAAGAACAATCT

GAAGGGGCGGCTGCATTTGGAGAGTCCTTCCCCCGGCGTGTACCGTCTCTTCATCCAGAACGTGGCTGTG

CAGGACAGCGGGACCTACAGCTGCCATGTGGAGGAGTGGCTGCCCAGCCCCAGTGGCATGTGGTATAAGC

GGGCAGAGGACACCGCTGGGCAGACAGCTCTGACAGTCATGCGACCAGATGCTTCCCTGCAGGTGGACAC

AGTGGTCCCCAATGCCACGGTCTCTGAGAAGGCAGCTTTCCAGCTGGACTGTAGCATCGTGTCCCGCTCC

AGCCAGGACTCCCGCTTCGCTGTGGCCTGGTATTCCCTGAGGACTAAAGCTGGGGGGAAAAGGAGCAGCC

CTGGCCTGGAAGAACAGGAAGAGGAAAGGGAGGAGGAGGAGGAGGAGGACGACGACGACGACGACGACCC

AACAGAGCGGACGGCCCTGCTGAGCGTGGGCCCAGATGCTGTCTTTGGCCCAGAGGGCAGTCCTTGGGAG

GGCAGGCTTCGCTTCCAGAGGCTCTCCCCGGTGCTCTACCGGCTCACAGTGCTGCAGGCAAGCCCCCAAG

ATACAGGCAATTACTCCTGCCATGTGGAGGAGTGGCTGCCCAGCCCTCAGAAGGAATGGTACCGGCTGAC

GGAGGAGGAGTCAGCCCCCATCGGCATCCGTGTTCTAGATACAAGTCCCACCCTCCAGTCCATCATCTGC

TCCAACGACGCACTCTTCTACTTCGTCTTCTTCTACCCTTTCCCCATCTTTGGCATTCTTATCATCACCA

TCCTTCTGGTGCGTTTCAAGAGCCGGAACTCCAGCAAGAACTCTGATGGGAAGAATGGGGTGCCTCTGCT

GTGGATCAAAGAGCCACACCTCAACTACTCCCCTACTTGCCTGGAGCCCCCTGTTCTCAGTATCCATCCA

GGGGCCATAGACTAA

 

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.

 

 

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.