Human JAG1/CD339 /AGS1/Jagged1 Lentivirus, Full-length Gene Clone in Lentivector, Pre-packaged Lentiviral Particles

Description

JAG1 (Jagged1) or CD339 is a 1218 amino acid (aa) single pass type I transmembrane glycoprotein that is the human homolog of the Drosophilia jagged protein. Human JAG1 is the ligand for the receptor notch 1, the latter a human homolog of the Drosophilia jagged receptor NOTCH.  The Notch signaling pathway is an intercellular signaling mechanism that is essential for proper embryonic development and cell fate decisions.  JAG1 interacts with NOTCH2 and NOTCH3, and with NOTCH1 in the presence of calcium ions.  JAG1 contains an N-terminal signal peptide, a 1034-aa extracellular domain (ECD) with a DSL domain and 16 EGF-like domains, a transmembrane domain (TM), and a 125-aa cytoplasmic tail.   JAG1 is involved in the mediation of Notch signaling. JAG1 may be involved in cell-fate decisions during hematopoiesis and in early and late stages of mammalian cardiovascular development. JAG1 inhibits myoblast differentiation and enhances fibroblast growth factor-induced angiogenesis.  JAG1 signalling through notch 1 has also been shown to play a role in hematopoiesis. Mutations in the JAG1 gene cause Alagille syndrome 1 (AGS1), an autosomal dominant multisystem disorder clinically defined by hepatic bile duct paucity and cholestasis in association with cardiac, skeletal, and ophthalmologic manifestations.

 

 

Gene Symbol: JAG1; CD339; AGS; AHD; AWS; HJ1; AGS1; JAGL1; Jagged1

 

NCBI Gene ID: 182

 

Uniprot Entry: P78504

 

Construct Details: Full length human JAG1 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: 3657 (bp)

 

Gene Insert Sequence:  

ATGCGTTCCCCACGGACGCGCGGCCGGTCCGGGCGCCCCCTAAGCCTCCTGCTCGCCCTGCTCTGTGCCC

TGCGAGCCAAGGTGTGTGGGGCCTCGGGTCAGTTCGAGTTGGAGATCCTGTCCATGCAGAACGTGAACGG

GGAGCTGCAGAACGGGAACTGCTGCGGCGGCGCCCGGAACCCGGGAGACCGCAAGTGCACCCGCGACGAG

TGTGACACATACTTCAAAGTGTGCCTCAAGGAGTATCAGTCCCGCGTCACGGCCGGGGGGCCCTGCAGCT

TCGGCTCAGGGTCCACGCCTGTCATCGGGGGCAACACCTTCAACCTCAAGGCCAGCCGCGGCAACGACCG

CAACCGCATCGTGCTGCCTTTCAGTTTCGCCTGGCCGAGGTCCTATACGTTGCTTGTGGAGGCGTGGGAT

TCCAGTAATGACACCGTTCAACCTGACAGTATTATTGAAAAGGCTTCTCACTCGGGCATGATCAACCCCA

GCCGGCAGTGGCAGACGCTGAAGCAGAACACGGGCGTTGCCCACTTTGAGTATCAGATCCGCGTGACCTG

TGATGACTACTACTATGGCTTTGGCTGCAATAAGTTCTGCCGCCCCAGAGATGACTTCTTTGGACACTAT

GCCTGTGACCAGAATGGCAACAAAACTTGCATGGAAGGCTGGATGGGCCCCGAATGTAACAGAGCTATTT

GCCGACAAGGCTGCAGTCCTAAGCATGGGTCTTGCAAACTCCCAGGTGACTGCAGGTGCCAGTACGGCTG

GCAAGGCCTGTACTGTGATAAGTGCATCCCACACCCGGGATGCGTCCACGGCATCTGTAATGAGCCCTGG

CAGTGCCTCTGTGAGACCAACTGGGGCGGCCAGCTCTGTGACAAAGATCTCAATTACTGTGGGACTCATC

AGCCGTGTCTCAACGGGGGAACTTGTAGCAACACAGGCCCTGACAAATATCAGTGTTCCTGCCCTGAGGG

GTATTCAGGACCCAACTGTGAAATTGCTGAGCACGCCTGCCTCTCTGATCCCTGTCACAACAGAGGCAGC

TGTAAGGAGACCTCCCTGGGCTTTGAGTGTGAGTGTTCCCCAGGCTGGACCGGCCCCACATGCTCTACAA

ACATTGATGACTGTTCTCCTAATAACTGTTCCCACGGGGGCACCTGCCAGGACCTGGTTAACGGATTTAA

GTGTGTGTGCCCCCCACAGTGGACTGGGAAAACGTGCCAGTTAGATGCAAATGAATGTGAGGCCAAACCT

TGTGTAAACGCCAAATCCTGTAAGAATCTCATTGCCAGCTACTACTGCGACTGTCTTCCCGGCTGGATGG

GTCAGAATTGTGACATAAATATTAATGACTGCCTTGGCCAGTGTCAGAATGACGCCTCCTGTCGGGATTT

GGTTAATGGTTATCGCTGTATCTGTCCACCTGGCTATGCAGGCGATCACTGTGAGAGAGACATCGATGAA

TGTGCCAGCAACCCCTGTTTGAATGGGGGTCACTGTCAGAATGAAATCAACAGATTCCAGTGTCTGTGTC

CCACTGGTTTCTCTGGAAACCTCTGTCAGCTGGACATCGATTATTGTGAGCCTAATCCCTGCCAGAACGG

TGCCCAGTGCTACAACCGTGCCAGTGACTATTTCTGCAAGTGCCCCGAGGACTATGAGGGCAAGAACTGC

TCACACCTGAAAGACCACTGCCGCACGACCCCCTGTGAAGTGATTGACAGCTGCACAGTGGCCATGGCTT

CCAACGACACACCTGAAGGGGTGCGGTATATTTCCTCCAACGTCTGTGGTCCTCACGGGAAGTGCAAGAG

TCAGTCGGGAGGCAAATTCACCTGTGACTGTAACAAAGGCTTCACGGGAACATACTGCCATGAAAATATT

AATGACTGTGAGAGCAACCCTTGTAGAAACGGTGGCACTTGCATCGATGGTGTCAACTCCTACAAGTGCA

TCTGTAGTGACGGCTGGGAGGGGGCCTACTGTGAAACCAATATTAATGACTGCAGCCAGAACCCCTGCCA

CAATGGGGGCACGTGTCGCGACCTGGTCAATGACTTCTACTGTGACTGTAAAAATGGGTGGAAAGGAAAG

ACCTGCCACTCACGTGACAGTCAGTGTGATGAGGCCACGTGCAACAACGGTGGCACCTGCTATGATGAGG

GGGATGCTTTTAAGTGCATGTGTCCTGGCGGCTGGGAAGGAACAACCTGTAACATAGCCCGAAACAGTAG

CTGCCTGCCCAACCCCTGCCATAATGGGGGCACATGTGTGGTCAACGGCGAGTCCTTTACGTGCGTCTGC

AAGGAAGGCTGGGAGGGGCCCATCTGTGCTCAGAATACCAATGACTGCAGCCCTCATCCCTGTTACAACA

GCGGCACCTGTGTGGATGGAGACAACTGGTACCGGTGCGAATGTGCCCCGGGTTTTGCTGGGCCCGACTG

CAGAATAAACATCAATGAATGCCAGTCTTCACCTTGTGCCTTTGGAGCGACCTGTGTGGATGAGATCAAT

GGCTACCGGTGTGTCTGCCCTCCAGGGCACAGTGGTGCCAAGTGCCAGGAAGTTTCAGGGAGACCTTGCA

TCACCATGGGGAGTGTGATACCAGATGGGGCCAAATGGGATGATGACTGTAATACCTGCCAGTGCCTGAA

TGGACGGATCGCCTGCTCAAAGGTCTGGTGTGGCCCTCGACCTTGCCTGCTCCACAAAGGGCACAGCGAG

TGCCCCAGCGGGCAGAGCTGCATCCCCATCCTGGACGACCAGTGCTTCGTCCACCCCTGCACTGGTGTGG

GCGAGTGTCGGTCTTCCAGTCTCCAGCCGGTGAAGACAAAGTGCACCTCTGACTCCTATTACCAGGATAA

CTGTGCGAACATCACATTTACCTTTAACAAGGAGATGATGTCACCAGGTCTTACTACGGAGCACATTTGC

AGTGAATTGAGGAATTTGAATATTTTGAAGAATGTTTCCGCTGAATATTCAATCTACATCGCTTGCGAGC

CTTCCCCTTCAGCGAACAATGAAATACATGTGGCCATTTCTGCTGAAGATATACGGGATGATGGGAACCC

GATCAAGGAAATCACTGACAAAATAATCGATCTTGTTAGTAAACGTGATGGAAACAGCTCGCTGATTGCT

GCCGTTGCAGAAGTAAGAGTTCAGAGGCGGCCTCTGAAGAACAGAACAGATTTCCTTGTTCCCTTGCTGA

GCTCTGTCTTAACTGTGGCTTGGATCTGTTGCTTGGTGACGGCCTTCTACTGGTGCCTGCGGAAGCGGCG

GAAGCCGGGCAGCCACACACACTCAGCCTCTGAGGACAACACCACCAACAACGTGCGGGAGCAGCTGAAC

CAGATCAAAAACCCCATTGAGAAACATGGGGCCAACACGGTCCCCATCAAGGATTATGAGAACAAGAACT

CCAAAATGTCTAAAATAAGGACACACAATTCTGAAGTAGAAGAGGACGACATGGACAAACACCAGCAGAA

AGCCCGGTTTGCCAAGCAGCCGGCGTACACGCTGGTAGACAGAGAAGAGAAGCCCCCCAACGGCACGCCG

ACAAAACACCCAAACTGGACAAACAAACAGGACAACAGAGACTTGGAAAGTGCCCAGAGCTTAAACCGAA

TGGAGTACATCGTATAG

 

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.

 

 

The product is shipped at 4°C for immediate use or with dry ice.  Upon receipt, centrifuge the vial briefly before opening.  Store at –80°C or lower and the product is stable for 3 months.  Avoid repeated freeze-thaw cycles.

 

 

The product should be employed in a Biosafety Level 2 tissue culture facility.

 

 

 

 

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

 

 

 

Additional supporting documents, including product data sheet (PDS), COA and MSDS are available upon request.

 

 

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