Human LEPR/CD295/OBR/LEPRD Lentivirus, Full-length Gene in Lentivector, Pre-packaged Lentiviral Particles

Description

CD295, OBR (OB receptor), or LEPR (leptin receptor) is a single pass type I transmembrane glycoprotein of the type I cytokine receptor family, type 2 subfamily.  LEPR has an extracellular region with 4 fibronectin type III (FN3) domains, 1 Immunoglobulin (Ig)-like domain, and a juxtamembrane WSXWS motif, a transmembrane domain (TM), and a cytoplasmic tail with a box 1 motif.  The WSXWS motif is necessary for proper protein folding and efficient intracellular transport and cell-surface receptor binding, while the box 1 motif is required for JAK interaction and activation. LEPR acts as a receptor for leptin (an adipocyte-specific hormone that regulates body weight), and is involved in the regulation of fat metabolism, as well as in a novel hematopoietic pathway that is required for normal lymphopoiesis. Upon ligand binding, LEPR mediates leptin central and peripheral effects through the activation of different signaling pathways such as JAK2/STAT3 and MAPK cascade. In the hypothalamus, leptin acts as an appetite-regulating factor that induces a decrease in food intake and an increase in energy consumption by inducing anorexinogenic factors and suppressing orexigenic neuropeptides, also regulates bone mass and secretion of hypothalamo-pituitary-adrenal hormones. In the periphery, leptin increases basal metabolism, influences reproductive function, regulates pancreatic beta-cell function and insulin secretion, is pro-angiogenic and affects innate and adaptive immunity. LEPR has a specific effect on T lymphocyte responses, differentially regulating the proliferation of naive and memory T -ells. Leptin increases Th1 and suppresses Th2 cytokine production. Mutations in the LEPR gene are associated with obesity and pituitary dysfunction. 

 

 

Gene Symbol: LEPR; CD295; OBR; OB-R; LEP-R; LEPRD; DB; HuB219

 

NCBI Gene ID: 3953

 

Uniprot Entry: P48357

 

Construct Details: Full length human LEPR/CD295 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, see the vector map above)

  

Gene Insert Size: 3498 (bp)

 

Gene Insert Sequence:  

ATGATTTGTCAAAAATTCTGTGTGGTTTTGTTACATTGGGAATTTATTTATGTGATAACTGCGTTTAACT

TGTCATATCCAATTACTCCTTGGAGATTTAAGTTGTCTTGCATGCCACCAAATTCAACCTATGACTACTT

CCTTTTGCCTGCTGGACTCTCAAAGAATACTTCAAATTCGAATGGACATTATGAGACAGCTGTTGAACCT

AAGTTTAATTCAAGTGGTACTCACTTTTCTAACTTATCCAAAACAACTTTCCACTGTTGCTTTCGGAGTG

AGCAAGATAGAAACTGCTCCTTATGTGCAGACAACATTGAAGGAAAGACATTTGTTTCAACAGTAAATTC

TTTAGTTTTTCAACAAATAGATGCAAACTGGAACATACAGTGCTGGCTAAAAGGAGACTTAAAATTATTC

ATCTGTTATGTGGAGTCATTATTTAAGAATCTATTCAGGAATTATAACTATAAGGTCCATCTTTTATATG

TTCTGCCTGAAGTGTTAGAAGATTCACCTCTGGTTCCCCAAAAAGGCAGTTTTCAGATGGTTCACTGCAA

TTGCAGTGTTCATGAATGTTGTGAATGTCTTGTGCCTGTGCCAACAGCCAAACTCAACGACACTCTCCTT

ATGTGTTTGAAAATCACATCTGGTGGAGTAATTTTCCAGTCACCTCTAATGTCAGTTCAGCCCATAAATA

TGGTGAAGCCTGATCCACCATTAGGTTTGCATATGGAAATCACAGATGATGGTAATTTAAAGATTTCTTG

GTCCAGCCCACCATTGGTACCATTTCCACTTCAATATCAAGTGAAATATTCAGAGAATTCTACAACAGTT

ATCAGAGAAGCTGACAAGATTGTCTCAGCTACATCCCTGCTAGTAGACAGTATACTTCCTGGGTCTTCGT

ATGAGGTTCAGGTGAGGGGCAAGAGACTGGATGGCCCAGGAATCTGGAGTGACTGGAGTACTCCTCGTGT

CTTTACCACACAAGATGTCATATACTTTCCACCTAAAATTCTGACAAGTGTTGGGTCTAATGTTTCTTTT

CACTGCATCTATAAGAAGGAAAACAAGATTGTTCCCTCAAAAGAGATTGTTTGGTGGATGAATTTAGCTG

AGAAAATTCCTCAAAGCCAGTATGATGTTGTGAGTGATCATGTTAGCAAAGTTACTTTTTTCAATCTGAA

TGAAACCAAACCTCGAGGAAAGTTTACCTATGATGCAGTGTACTGCTGCAATGAACATGAATGCCATCAT

CGCTATGCTGAATTATATGTGATTGATGTCAATATCAATATCTCATGTGAAACTGATGGGTACTTAACTA

AAATGACTTGCAGATGGTCAACCAGTACAATCCAGTCACTTGCGGAAAGCACTTTGCAATTGAGGTATCA

TAGGAGCAGCCTTTACTGTTCTGATATTCCATCTATTCATCCCATATCTGAGCCCAAAGATTGCTATTTG

CAGAGTGATGGTTTTTATGAATGCATTTTCCAGCCAATCTTCCTATTATCTGGCTACACAATGTGGATTA

GGATCAATCACTCTCTAGGTTCACTTGACTCTCCACCAACATGTGTCCTTCCTGATTCTGTGGTGAAGCC

ACTGCCTCCATCCAGTGTGAAAGCAGAAATTACTATAAACATTGGATTATTGAAAATATCTTGGGAAAAG

CCAGTCTTTCCAGAGAATAACCTTCAATTCCAGATTCGCTATGGTTTAAGTGGAAAAGAAGTACAATGGA

AGATGTATGAGGTTTATGATGCAAAATCAAAATCTGTCAGTCTCCCAGTTCCAGACTTGTGTGCAGTCTA

TGCTGTTCAGGTGCGCTGTAAGAGGCTAGATGGACTGGGATATTGGAGTAATTGGAGCAATCCAGCCTAC

ACAGTTGTCATGGATATAAAAGTTCCTATGAGAGGACCTGAATTTTGGAGAATAATTAATGGAGATACTA

TGAAAAAGGAGAAAAATGTCACTTTACTTTGGAAGCCCCTGATGAAAAATGACTCATTGTGCAGTGTTCA

GAGATATGTGATAAACCATCATACTTCCTGCAATGGAACATGGTCAGAAGATGTGGGAAATCACACGAAA

TTCACTTTCCTGTGGACAGAGCAAGCACATACTGTTACGGTTCTGGCCATCAATTCAATTGGTGCTTCTG

TTGCAAATTTTAATTTAACCTTTTCATGGCCTATGAGCAAAGTAAATATCGTGCAGTCACTCAGTGCTTA

TCCTTTAAACAGCAGTTGTGTGATTGTTTCCTGGATACTATCACCCAGTGATTACAAGCTAATGTATTTT

ATTATTGAGTGGAAAAATCTTAATGAAGATGGTGAAATAAAATGGCTTAGAATCTCTTCATCTGTTAAGA

AGTATTATATCCATGATCATTTTATCCCCATTGAGAAGTACCAGTTCAGTCTTTACCCAATATTTATGGA

AGGAGTGGGAAAACCAAAGATAATTAATAGTTTCACTCAAGATGATATTGAAAAACACCAGAGTGATGCA

GGTTTATATGTAATTGTGCCAGTAATTATTTCCTCTTCCATCTTATTGCTTGGAACATTATTAATATCAC

ACCAAAGAATGAAAAAGCTATTTTGGGAAGATGTTCCGAACCCCAAGAATTGTTCCTGGGCACAAGGACT

TAATTTTCAGAAGCCAGAAACGTTTGAGCATCTTTTTATCAAGCATACAGCATCAGTGACATGTGGTCCT

CTTCTTTTGGAGCCTGAAACAATTTCAGAAGATATCAGTGTTGATACATCATGGAAAAATAAAGATGAGA

TGATGCCAACAACTGTGGTCTCTCTACTTTCAACAACAGATCTTGAAAAGGGTTCTGTTTGTATTAGTGA

CCAGTTCAACAGTGTTAACTTCTCTGAGGCTGAGGGTACTGAGGTAACCTATGAGGACGAAAGCCAGAGA

CAACCCTTTGTTAAATACGCCACGCTGATCAGCAACTCTAAACCAAGTGAAACTGGTGAAGAACAAGGGC

TTATAAATAGTTCAGTCACCAAGTGCTTCTCTAGCAAAAATTCTCCGTTGAAGGATTCTTTCTCTAATAG

CTCATGGGAGATAGAGGCCCAGGCATTTTTTATATTATCAGATCAGCATCCCAACATAATTTCACCACAC

CTCACATTCTCAGAAGGATTGGATGAACTTTTGAAATTGGAGGGAAATTTCCCTGAAGAAAATAATGATA

AAAAGTCTATCTATTATTTAGGGGTCACCTCAATCAAAAAGAGAGAGAGTGGTGTGCTTTTGACTGACAA

GTCAAGGGTATCGTGCCCATTCCCAGCCCCCTGTTTATTCACGGACATCAGAGTTCTCCAGGACAGTTGC

TCACACTTTGTAGAAAATAATATCAACTTAGGAACTTCTAGTAAGAAGACTTTTGCATCTTACATGCCTC

AATTCCAAACTTGTTCTACTCAGACTCATAAGATCATGGAAAACAAGATGTGTGACCTAACTGTGTAA

 

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.

 

 

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

 

 

 

 

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.