NGFR (nerve growth factor receptor), also known as Low affinity neurotrophin receptor p75NTR and CD271, is a single-pass type I transmembrane glycoprotein in the TNF receptor superfamily (TNFRSF16).  The extracellular region contains four cysteine­rich domains and binds NGF, BDNF, NT­3 and NT­4 approximately equally with low affinity. The cytoplasmic region contains a death domain (DD). NGFR is widely expressed during development and in the adult. Expression has been detected in both neuronal and non­neuronal cells. NGFR was originally reported to function as a positive regulator of TrkA, a high- affinity nerve growth factor receptor. NGFR has also been shown to signal by itself. Depending on its cellular environment, NGFR has now been shown to regulate cell migration, gene expression and to mediate apoptosis. Recombinant NGF R/Fc chimera binds NGF with high affinity and is a potent NGF antagonist. Naturally occurring truncated NGF R containing the extracellular domain and lacking the transmembrane or intracellular domain has been detected in vivo in urine, plasma and in amniotic fluid of humans and animals. NGFR is also necessary for the circadian oscillation of the clock genes in the suprachiasmatic nucleus of the brain and in liver and of the genes involved in glucose and lipid metabolism in the liver. It may plays a role in the regulation of the translocation of GLUT4 to the cell surface in adipocytes and skeletal muscle cells in response to insulin and thereby contributes to the regulation of insulin-dependent glucose uptake.

NGFR (nerve growth factor receptor), also known as Low affinity neurotrophin receptor p75NTR and CD271, is a single-pass type I transmembrane glycoprotein in the TNF receptor superfamily (TNFRSF16).  The extracellular region contains four cysteine­rich domains and binds NGF, BDNF, NT­3 and NT­4 approximately equally with low affinity. The cytoplasmic region contains a death domain (DD). NGFR is widely expressed during development and in the adult. Expression has been detected in both neuronal and non­neuronal cells. NGFR was originally reported to function as a positive regulator of TrkA, a high- affinity nerve growth factor receptor. NGFR has also been shown to signal by itself. Depending on its cellular environment, NGFR has now been shown to regulate cell migration, gene expression and to mediate apoptosis. Recombinant NGF R/Fc chimera binds NGF with high affinity and is a potent NGF antagonist. Naturally occurring truncated NGF R containing the extracellular domain and lacking the transmembrane or intracellular domain has been detected in vivo in urine, plasma and in amniotic fluid of humans and animals. NGFR is also necessary for the circadian oscillation of the clock genes in the suprachiasmatic nucleus of the brain and in liver and of the genes involved in glucose and lipid metabolism in the liver. It may plays a role in the regulation of the translocation of GLUT4 to the cell surface in adipocytes and skeletal muscle cells in response to insulin and thereby contributes to the regulation of insulin-dependent glucose uptake.

  

Product Details

 

Gene Symbol: TNFRSF16; CD271; p75NTR; p75(NTR); Gp80-LNGFR; p75 ICD

  

NCBI Gene ID: 4804

 

Uniprot Entry: P08138

 

Construct Details: Full length human NGFR gene is subcloned into the Lentiviral expression vector pLTC with an upstream CMV promoter, 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.

 

Vector Type: pLTC (lentiviral expression vector containing a heterologous CMV promoter, see the vector map above)

 

Gene Insert Sequence:  

ATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGG

TGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGG

CCCAGCCTTGTGGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACC

GAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGAGGCCGACGACGCCGTGTG

CCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCC

TCGTGTTCTCCTGCCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACCAC

GTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGTGCACACGCTGGGCCGACGCCGA

GTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGG

AGCCTGAGGCACCTCCAGAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAG

CCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGGCCTTGT

GGCCTACATAGCCTTCAAGAGGTGGAACAGCTGCAAGCAGAACAAGCAAGGAGCCAACAGCCGGCCAGTGAACCAGACGC

CCCCACCAGAGGGAGAAAAACTCCACAGCGACAGTGGCATCTCCGTGGACAGCCAGAGCCTGCATGACCAGCAGCCCCAC

ACGCAGACAGCCTCGGGCCAGGCCCTCAAGGGTGACGGAGGCCTCTACAGCAGCCTGCCCCCAGCCAAGCGGGAGGAGGT

GGAGAAGCTTCTCAACGGCTCTGCGGGGGACACCTGGCGGCACCTGGCGGGCGAGCTGGGCTACCAGCCCGAGCACATAG

ACTCCTTTACCCATGAGGCCTGCCCCGTTCGCGCCCTGCTTGCAAGCTGGGCCACCCAGGACAGCGCCACACTGGACGCC

CTCCTGGCCGCCCTGCGCCGCATCCAGCGAGCCGACCTCGTGGAGAGTCTGTGCAGTGAGTCCACTGCCACATCCCCGGT

GTGA

 

Primers: Gene insert coding sequence can be confirmed by following primers:

(a) forward (or 5'-end) primer: 5’-CAAATGGGCGGTAGGCGTG-3’;

(b) reverse (or 3'-end) primer: 5’-CGGGAAGCAATAGCATGATA-3’

 

 

Note: The sequence may vary due to the naturally occurring variants or mutations, such as single nucleotide polymorphism, or the artifact during PCR amplification.  High fidelity PCR systems should always be used. 

 

 

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”.

 

Troubleshooting

 

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.

 

Storage

 

The product is shipped at 4°C for immediate use.  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.

 

References

 

1. Cell 47:545-554 (1986).

2. J. Cell Biol. 140:911 (1998).

3. J Biol Chem. 279 (3): 1703 (2004).

4. Mol Biol Cell. 20 (16): 3620 (2009).

5. J. Neurosci. 33:10221 (2013). 

 

 

 

Documentation

 

Product datasheet (pdf) can be downloaded here: LTV0104-PDS.pdf

 

 

 

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 produ