Human CASR/CAR/PCaR1/GPRC2A Lentivirus, Full-length Gene in Lentivector, Pre-packaged Lentiviral Particles

Description

CASR (calcium sensing receptor) is a 7 transmembrane (7TM) receptor belonging to the family 3 (also known as family C) of the G-protein-coupled receptors (GPCRs). Structurally they are composed of 4 elements; an N-terminal signal peptide; a large hydrophilic extracellular agonist-binding region; a region containing 7TM domains; and a C-terminal cytoplasmic domain of variable length. Family 3 members include the metabotropic glutamate receptors (mGluRs), the extracellular calcium-sensing receptors (CASRs), the gamma-amino-butyric acid (GABA) type B receptors, and the vomeronasal type-2 receptors.  These GPCRs regulate many important physiological processes and many of them are promising targets for drug development. CASR is expressed in the parathyroid hormone (PTH)-producing chief cells of the parathyroid gland, and the cells lining the kidney tubule.  CASR senses small changes in circulating calcium concentration and couples this information to intracellular signaling pathways that activate a phosphatidylinositol-calcium second messenger system and modify PTH secretion or renal cation handling, thus this protein plays an essential role in maintaining mineral ion homeostasis. Mutations in the CASR gene cause familial hypocalciuric hypercalcemia, familial, isolated hypoparathyroidism, and neonatal severe primary hyperparathyroidism. 

 

 

Gene Symbol: CASR; CAR; FHH; FIH; HHC; EIG8; HHC1; NSHPT; PCAR1; GPRC2A; HYPOC1

 

NCBI Gene ID: 846

 

Uniprot Entry: P41180

 

Construct Details: Full length human CASR 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: 3237 (bp)

 

Gene Insert Sequence:  

ATGGCATTTTATAGCTGCTGCTGGGTCCTCTTGGCACTCACCTGGCACACCTCTGCCTACGGGCCAGACC

AGCGAGCCCAAAAGAAGGGGGACATTATCCTTGGGGGGCTCTTTCCTATTCATTTTGGAGTAGCAGCTAA

AGATCAAGATCTCAAATCAAGGCCGGAGTCTGTGGAATGTATCAGGTATAATTTCCGTGGGTTTCGCTGG

TTACAGGCTATGATATTTGCCATAGAGGAGATAAACAGCAGCCCAGCCCTTCTTCCCAACTTGACGCTGG

GATACAGGATATTTGACACTTGCAACACCGTTTCTAAGGCCTTGGAAGCCACCCTGAGTTTTGTTGCTCA

AAACAAAATTGATTCTTTGAACCTTGATGAGTTCTGCAACTGCTCAGAGCACATTCCCTCTACGATTGCT

GTGGTGGGAGCAACTGGCTCAGGCGTCTCCACGGCAGTGGCAAATCTGCTGGGGCTCTTCTACATTCCCC

AGGTCAGTTATGCCTCCTCCAGCAGACTCCTCAGCAACAAGAATCAATTCAAGTCTTTCCTCCGAACCAT

CCCCAATGATGAGCACCAGGCCACTGCCATGGCAGACATCATCGAGTATTTCCGCTGGAACTGGGTGGGC

ACAATTGCAGCTGATGACGACTATGGGCGGCCGGGGATTGAGAAATTCCGAGAGGAAGCTGAGGAAAGGG

ATATCTGCATCGACTTCAGTGAACTCATCTCCCAGTACTCTGATGAGGAAGAGATCCAGCATGTGGTAGA

GGTGATTCAAAATTCCACGGCCAAAGTCATCGTGGTTTTCTCCAGTGGCCCAGATCTTGAGCCCCTCATC

AAGGAGATTGTCCGGCGCAATATCACGGGCAAGATCTGGCTGGCCAGCGAGGCCTGGGCCAGCTCCTCCC

TGATCGCCATGCCTCAGTACTTCCACGTGGTTGGCGGCACCATTGGATTCGCTCTGAAGGCTGGGCAGAT

CCCAGGCTTCCGGGAATTCCTGAAGAAGGTCCATCCCAGGAAGTCTGTCCACAATGGTTTTGCCAAGGAG

TTTTGGGAAGAAACATTTAACTGCCACCTCCAAGAAGGTGCAAAAGGACCTTTACCTGTGGACACCTTTC

TGAGAGGTCACGAAGAAAGTGGCGACAGGTTTAGCAACAGCTCGACAGCCTTCCGACCCCTCTGTACAGG

GGATGAGAACATCAGCAGTGTCGAGACCCCTTACATAGATTACACGCATTTACGGATATCCTACAATGTG

TACTTAGCAGTCTACTCCATTGCCCACGCCTTGCAAGATATATATACCTGCTTACCTGGGAGAGGGCTCT

TCACCAATGGCTCCTGTGCAGACATCAAGAAAGTTGAGGCGTGGCAGGTCCTGAAGCACCTACGGCATCT

AAACTTTACAAACAATATGGGGGAGCAGGTGACCTTTGATGAGTGTGGTGACCTGGTGGGGAACTATTCC

ATCATCAACTGGCACCTCTCCCCAGAGGATGGCTCCATCGTGTTTAAGGAAGTCGGGTATTACAACGTCT

ATGCCAAGAAGGGAGAAAGACTCTTCATCAACGAGGAGAAAATCCTGTGGAGTGGGTTCTCCAGGGAGGT

GCCCTTCTCCAACTGCAGCCGAGACTGCCTGGCAGGGACCAGGAAAGGGATCATTGAGGGGGAGCCCACC

TGCTGCTTTGAGTGTGTGGAGTGTCCTGATGGGGAGTATAGTGATGAGACAGATGCCAGTGCCTGTAACA

AGTGCCCAGATGACTTCTGGTCCAATGAGAACCACACCTCCTGCATTGCCAAGGAGATCGAGTTTCTGTC

GTGGACGGAGCCCTTTGGGATCGCACTCACCCTCTTTGCCGTGCTGGGCATTTTCCTGACAGCCTTTGTG

CTGGGTGTGTTTATCAAGTTCCGCAACACACCCATTGTCAAGGCCACCAACCGAGAGCTCTCCTACCTCC

TCCTCTTCTCCCTGCTCTGCTGCTTCTCCAGCTCCCTGTTCTTCATCGGGGAGCCCCAGGACTGGACGTG

CCGCCTGCGCCAGCCGGCCTTTGGCATCAGCTTCGTGCTCTGCATCTCATGCATCCTGGTGAAAACCAAC

CGTGTCCTCCTGGTGTTTGAGGCCAAGATCCCCACCAGCTTCCACCGCAAGTGGTGGGGGCTCAACCTGC

AGTTCCTGCTGGTTTTCCTCTGCACCTTCATGCAGATTGTCATCTGTGTGATCTGGCTCTACACCGCGCC

CCCGTCAAGCTACCGCAACCAGGAGCTGGAGGATGAGATCATCTTCATCACGTGCCACGAGGGCTCCCTC

ATGGCCCTGGGCTTCCTGATCGGCTACACCTGCCTGCTGGCTGCCATCTGCTTCTTCTTTGCCTTCAAGT

CCCGGAAGCTGCCGGAGAACTTCAATGAAGCCAAGTTCATCACCTTCAGCATGCTCATCTTCTTCATCGT

CTGGATCTCCTTCATTCCAGCCTATGCCAGCACCTATGGCAAGTTTGTCTCTGCCGTAGAGGTGATTGCC

ATCCTGGCAGCCAGCTTTGGCTTGCTGGCGTGCATCTTCTTCAACAAGATCTACATCATTCTCTTCAAGC

CATCCCGCAACACCATCGAGGAGGTGCGTTGCAGCACCGCAGCTCACGCTTTCAAGGTGGCTGCCCGGGC

CACGCTGCGCCGCAGCAACGTCTCCCGCAAGCGGTCCAGCAGCCTTGGAGGCTCCACGGGATCCACCCCC

TCCTCCTCCATCAGCAGCAAGAGCAACAGCGAAGACCCATTCCCACAGCCCGAGAGGCAGAAGCAGCAGC

AGCCGCTGGCCCTAACCCAGCAAGAGCAGCAGCAGCAGCCCCTGACCCTCCCACAGCAGCAACGATCTCA

GCAGCAGCCCAGATGCAAGCAGAAGGTCATCTTTGGCAGCGGCACGGTCACCTTCTCACTGAGCTTTGAT

GAGCCTCAGAAGAACGCCATGGCCCACAGGAATTCTACGCACCAGAACTCCCTGGAGGCCCAGAAAAGCA

GCGATACGCTGACCCGACACGAGCCATTACTCCCGCTGCAGTGCGGGGAAACGGACTTAGATCTGACCGT

CCAGGAAACAGGTCTGCAAGGACCTGTGGGTGGAGACCAGCGGCCAGAGGTGGAGGACCCTGAAGAGTTG

TCCCCAGCACTTGTAGTGTCCAGTTCACAGAGCTTTGTCATCAGTGGTGGAGGCAGCACTGTTACAGAAA

ACGTAGTGAATTCATAA

 

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

 

 

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”.