Human NTRK2/TRKB/OBHD Lentivirus, Full-length Gene in Lentivector, Pre-packaged Lentiviral Particles

Description

NTRK2 (neurotrophic receptor tyrosine kinase 2) receptor tyrosine kinase (RTK), is a single-pass type I transmembrane glycoprotein that belongs to the insulin receptor (InsR) subfamily of the tyrosine protein kinase family. NTRK2 comprises a 390-aa extracellular domain (ECD), a transmembrane region (TM), and a 368-aa intracellular kinase domain. The ECD contains 2 LRR (leucine-rich repeat) domains, 1 LRRCT and 2 Ig-like C2-type domains.  NTRK2 acts as a receptor for BDNF/brain-derived neurotrophic factor and NTF4/neurotrophin-4.  Upon neurotrophin binding, NTRK2 phosphorylates itself and members of the MAPK pathway. NTRK2 is involved in the development and the maturation of the central and the peripheral nervous systems through regulation of neuron survival, proliferation, migration, differentiation, and synapse formation and plasticity. NTRK2 plays a role in learning and memory by regulating both short term synaptic function and long-term potentiation and in in neutrophin-dependent calcium signaling in glial cells and mediate communication between neurons and glia. Mutations in the NTRK2  gene have been associated with obesity and mood disorders (OBHD). 

 

 

Gene Symbol: NTRK2; OBHD; TRKB; trk-B; GP145-TrkB

 

NCBI Gene ID: 4915

 

Uniprot Entry: Q16620

 

Construct Details: Full length human NTRK2 gene is subcloned into the Lentiviral expression vector pLTC with an upstream CMV promoter and with or without an antibiotic 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: 2469 (bp)

 

Gene Insert Sequence:  

ATGTCGTCCTGGATAAGGTGGCATGGACCCGCCATGGCGCGGCTCTGGGGCTTCTGCTGGCTGGTTGTGG

GCTTCTGGAGGGCCGCTTTCGCCTGTCCCACGTCCTGCAAATGCAGTGCCTCTCGGATCTGGTGCAGCGA

CCCTTCTCCTGGCATCGTGGCATTTCCGAGATTGGAGCCTAACAGTGTAGATCCTGAGAACATCACCGAA

ATTTTCATCGCAAACCAGAAAAGGTTAGAAATCATCAACGAAGATGATGTTGAAGCTTATGTGGGACTGA

GAAATCTGACAATTGTGGATTCTGGATTAAAATTTGTGGCTCATAAAGCATTTCTGAAAAACAGCAACCT

GCAGCACATCAATTTTACCCGAAACAAACTGACGAGTTTGTCTAGGAAACATTTCCGTCACCTTGACTTG

TCTGAACTGATCCTGGTGGGCAATCCATTTACATGCTCCTGTGACATTATGTGGATCAAGACTCTCCAAG

AGGCTAAATCCAGTCCAGACACTCAGGATTTGTACTGCCTGAATGAAAGCAGCAAGAATATTCCCCTGGC

AAACCTGCAGATACCCAATTGTGGTTTGCCATCTGCAAATCTGGCCGCACCTAACCTCACTGTGGAGGAA

GGAAAGTCTATCACATTATCCTGTAGTGTGGCAGGTGATCCGGTTCCTAATATGTATTGGGATGTTGGTA

ACCTGGTTTCCAAACATATGAATGAAACAAGCCACACACAGGGCTCCTTAAGGATAACTAACATTTCATC

CGATGACAGTGGGAAGCAGATCTCTTGTGTGGCGGAAAATCTTGTAGGAGAAGATCAAGATTCTGTCAAC

CTCACTGTGCATTTTGCACCAACTATCACATTTCTCGAATCTCCAACCTCAGACCACCACTGGTGCATTC

CATTCACTGTGAAAGGCAACCCCAAACCAGCGCTTCAGTGGTTCTATAACGGGGCAATATTGAATGAGTC

CAAATACATCTGTACTAAAATACATGTTACCAATCACACGGAGTACCACGGCTGCCTCCAGCTGGATAAT

CCCACTCACATGAACAATGGGGACTACACTCTAATAGCCAAGAATGAGTATGGGAAGGATGAGAAACAGA

TTTCTGCTCACTTCATGGGCTGGCCTGGAATTGACGATGGTGCAAACCCAAATTATCCTGATGTAATTTA

TGAAGATTATGGAACTGCAGCGAATGACATCGGGGACACCACGAACAGAAGTAATGAAATCCCTTCCACA

GACGTCACTGATAAAACCGGTCGGGAACATCTCTCGGTCTATGCTGTGGTGGTGATTGCGTCTGTGGTGG

GATTTTGCCTTTTGGTAATGCTGTTTCTGCTTAAGTTGGCAAGACACTCCAAGTTTGGCATGAAAGGCCC

AGCCTCCGTTATCAGCAATGATGATGACTCTGCCAGCCCACTCCATCACATCTCCAATGGGAGTAACACT

CCATCTTCTTCGGAAGGTGGCCCAGATGCTGTCATTATTGGAATGACCAAGATCCCTGTCATTGAAAATC

CCCAGTACTTTGGCATCACCAACAGTCAGCTCAAGCCAGACACATTTGTTCAGCACATCAAGCGACATAA

CATTGTTCTGAAAAGGGAGCTAGGCGAAGGAGCCTTTGGAAAAGTGTTCCTAGCTGAATGCTATAACCTC

TGTCCTGAGCAGGACAAGATCTTGGTGGCAGTGAAGACCCTGAAGGATGCCAGTGACAATGCACGCAAGG

ACTTCCACCGTGAGGCCGAGCTCCTGACCAACCTCCAGCATGAGCACATCGTCAAGTTCTATGGCGTCTG

CGTGGAGGGCGACCCCCTCATCATGGTCTTTGAGTACATGAAGCATGGGGACCTCAACAAGTTCCTCAGG

GCACACGGCCCTGATGCCGTGCTGATGGCTGAGGGCAACCCGCCCACGGAACTGACGCAGTCGCAGATGC

TGCATATAGCCCAGCAGATCGCCGCGGGCATGGTCTACCTGGCGTCCCAGCACTTCGTGCACCGCGATTT

GGCCACCAGGAACTGCCTGGTCGGGGAGAACTTGCTGGTGAAAATCGGGGACTTTGGGATGTCCCGGGAC

GTGTACAGCACTGACTACTACAGGGTCGGTGGCCACACAATGCTGCCCATTCGCTGGATGCCTCCAGAGA

GCATCATGTACAGGAAATTCACGACGGAAAGCGACGTCTGGAGCCTGGGGGTCGTGTTGTGGGAGATTTT

CACCTATGGCAAACAGCCCTGGTACCAGCTGTCAAACAATGAGGTGATAGAGTGTATCACTCAGGGCCGA

GTCCTGCAGCGACCCCGCACGTGCCCCCAGGAGGTGTATGAGCTGATGCTGGGGTGCTGGCAGCGAGAGC

CCCACATGAGGAAGAACATCAAGGGCATCCATACCCTCCTTCAGAACTTGGCCAAGGCATCTCCGGTCTA

CCTGGACATTCTAGGCTAG

 

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.

 

 

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 PDS, COA and MSDS are available upon request.

 

 

 

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