Human INSRR/IRR Lentivirus, Full-length Gene in Lentivector, Pre-packaged Lentiviral Particles

Description

INSRR (insulin receptor-related protein) 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.  INSRR is expressed as a 1297-aa precursor and cleavage of the precursor generates alpha and beta subunits. The alpha subunit comprises a 716-aa extracellular domain (ECD) while the beta subunit contains a 175-aa extracellular domain (ECD), a transmembrane region (TM), and a 354-aa intracellular kinase domain. IINSRR probably exist as a tetramer of 2 alpha and 2 beta chains linked by disulfide bonds. The alpha chains contribute to the formation of the ligand-binding domain, while the beta chain carries the kinase domain for signaling. INSRR may function as a pH sensing receptor which is activated by increased extracellular pH. INSRR activates an intracellular signaling pathway that involves IRS1 and AKT1/PKB.

 

 

Gene Symbol: INSRR; IRR

 

NCBI Gene ID: 3645

 

Uniprot Entry: P14616

 

Construct Details: Full length human INSRR 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: 3894 (bp)

 

Gene Insert Sequence:  

ATGGCAGTGCCTAGTCTGTGGCCCTGGGGAGCATGCCTGCCTGTGATCTTCCTCTCCTTGGGATTTGGCC

TGGATACAGTAGAGGTGTGCCCCAGCCTGGATATTCGCTCAGAGGTGGCAGAGCTTCGTCAGCTGGAGAA

CTGCAGCGTGGTGGAGGGCCACCTGCAGATCCTGCTCATGTTCACAGCCACCGGGGAGGACTTCCGCGGC

CTCAGCTTCCCTCGCCTCACCCAGGTCACCGACTACCTGCTGCTCTTCCGTGTCTACGGACTGGAGAGCC

TGCGCGACCTCTTCCCCAACCTAGCAGTCATCCGCGGGACGCGCCTCTTCCTGGGCTATGCACTGGTCAT

CTTTGAGATGCCACATCTGCGTGACGTGGCACTGCCTGCACTTGGGGCCGTGCTGCGTGGGGCTGTGCGT

GTGGAGAAGAACCAGGAGCTCTGCCACCTCTCCACCATTGACTGGGGACTGCTGCAGCCAGCACCTGGCG

CCAACCACATCGTGGGCAACAAGCTGGGCGAGGAGTGTGCTGACGTGTGCCCTGGTGTGCTGGGTGCTGC

TGGTGAGCCCTGTGCCAAGACCACCTTCAGCGGGCACACTGACTACAGATGCTGGACCTCCAGCCACTGC

CAGAGAGTGTGCCCCTGCCCCCATGGGATGGCTTGCACAGCGAGGGGCGAGTGCTGCCACACCGAATGCC

TGGGGGGCTGCAGCCAGCCAGAAGACCCTCGTGCCTGTGTAGCTTGCCGCCACCTCTACTTCCAGGGTGC

CTGCCTGTGGGCCTGCCCGCCAGGCACCTACCAGTATGAGTCCTGGCGCTGTGTCACAGCTGAGCGCTGT

GCCAGCCTGCACTCTGTGCCCGGCCGTGCCTCCACCTTCGGCATACACCAGGGCAGTTGCCTGGCCCAGT

GCCCTTCTGGCTTCACCCGTAATAGCAGCAGCATATTCTGCCACAAGTGCGAGGGGCTGTGCCCTAAAGA

GTGCAAGGTAGGCACCAAGACCATCGACTCCATCCAGGCGGCACAGGATCTTGTGGGCTGCACGCATGTG

GAGGGAAGCCTCATCCTCAACCTTCGCCAGGGCTACAACCTGGAGCCACAGCTGCAGCACAGCCTGGGGC

TGGTAGAAACCATTACTGGCTTCCTCAAAATCAAGCACTCCTTTGCCCTCGTGTCCCTGGGCTTTTTCAA

GAACCTCAAACTAATCCGGGGAGACGCCATGGTGGATGGGAACTACACTCTCTACGTGCTGGACAACCAG

AACCTACAACAGCTAGGGTCCTGGGTGGCCGCGGGGCTCACCATTCCCGTGGGCAAGATCTACTTCGCCT

TCAACCCGCGCCTCTGCTTGGAACACATCTACCGACTGGAGGAGGTGACAGGCACGCGAGGTCGGCAGAA

CAAGGCTGAGATCAACCCCCGCACCAACGGAGACCGCGCCGCCTGCCAGACTCGCACCCTGCGCTTCGTG

TCCAACGTGACGGAGGCAGACCGCATCCTGCTACGCTGGGAGCGCTATGAGCCACTGGAGGCCCGCGACC

TGCTCAGCTTCATCGTGTACTACAAGGAGTCCCCATTCCAGAACGCCACAGAGCACGTGGGTCCAGATGC

TTGTGGAACCCAGAGCTGGAACCTGCTGGATGTGGAGCTGCCCCTAAGCCGCACCCAGGAGCCAGGGGTG

ACCCTAGCCTCCCTCAAGCCTTGGACACAGTACGCAGTGTTTGTGCGGGCCATCACGCTAACCACTGAGG

AGGACAGCCCTCATCAAGGAGCCCAGAGTCCCATCGTCTACCTCCGAACGCTGCCTGCAGCTCCCACGGT

GCCCCAAGACGTCATCTCCACGTCCAACTCCTCCTCCCACCTCCTGGTGCGCTGGAAGCCACCGACCCAG

CGCAATGGGAACCTCACCTACTACCTGGTGCTGTGGCAGCGGCTGGCAGAGGACGGCGACCTCTACCTCA

ATGACTACTGCCACCGCGGCTTGCGGCTGCCCACCAGCAACAACGATCCGCGCTTCGACGGCGAAGACGG

GGATCCTGAGGCCGAGATGGAGTCCGACTGCTGCCCTTGCCAGCACCCACCTCCTGGTCAGGTTCTGCCC

CCGCTGGAGGCGCAAGAGGCCTCGTTCCAGAAGAAGTTTGAAAACTTTCTACACAACGCGATCACCATCC

CCATATCCCCTTGGAAGGTGACGTCCATCAACAAGAGCCCCCAAAGGGACTCAGGGCGGCACCGCCGGGC

AGCTGGGCCCCTCCGGCTGGGGGGCAACAGCTCGGATTTCGAGATCCAGGAGGACAAGGTGCCCCGTGAG

CGAGCGGTGCTGAGCGGCCTGCGCCACTTCACGGAATACCGGATCGACATCCATGCCTGCAACCACGCGG

CGCACACCGTGGGCTGCAGCGCCGCCACCTTCGTCTTTGCGCGCACCATGCCCCACAGAGAGGCTGATGG

TATTCCAGGAAAGGTGGCCTGGGAGGCCTCCAGCAAGAACAGTGTCCTTCTGCGCTGGCTCGAGCCACCA

GACCCCAACGGACTCATCCTCAAGTACGAAATCAAGTACCGCCGCTTGGGAGAGGAGGCCACAGTGCTGT

GTGTGTCCCGTCTTCGATATGCGAAGTTTGGGGGAGTCCACCTGGCCCTGCTGCCCCCTGGAAACTACTC

TGCCAGGGTTAGGGCAACCTCACTGGCTGGCAATGGCTCTTGGACAGACAGTGTTGCCTTCTACATCCTT

GGCCCAGAGGAGGAGGATGCTGGGGGGCTGCATGTCCTCCTCACTGCCACCCCTGTGGGGCTCACGCTGC

TCATCGTTCTTGCTGCCCTTGGTTTCTTCTACGGCAAGAAGAGAAACAGAACCCTGTATGCTTCTGTGAA

TCCAGAGTACTTCAGCGCCTCTGATATGTATGTCCCTGATGAATGGGAGGTGCCTCGGGAGCAGATCTCG

ATAATCCGGGAACTGGGCCAGGGCTCTTTTGGGATGGTATATGAGGGGCTGGCACGAGGACTTGAGGCTG

GAGAGGAGTCCACACCCGTGGCCCTGAAGACGGTGAATGAGCTGGCCAGCCCACGGGAATGCATTGAGTT

CCTCAAGGAAGCTTCTGTCATGAAAGCCTTCAAGTGTCACCATGTGGTGCGTCTCCTGGGTGTGGTATCT

CAGGGCCAGCCAACTCTGGTCATCATGGAGTTAATGACCCGTGGGGACCTCAAGAGCCATCTTCGATCTT

TGCGGCCTGAGGCAGAGAACAACCCTGGGCTCCCACAGCCAGCATTGGGGGAAATGATCCAAATGGCTGG

TGAGATTGCAGACGGCATGGCCTACCTTGCTGCCAACAAGTTTGTGCACCGAGATCTAGCAGCCCGCAAC

TGCATGGTGTCCCAGGACTTCACCGTCAAGATCGGGGACTTCGGGATGACTCGGGACGTGTATGAGACAG

ACTATTACCGCAAGGGTGGGAAGGGGCTGCTGCCCGTGCGCTGGATGGCCCCCGAGTCCCTCAAAGATGG

GATCTTCACCACCCACTCGGATGTCTGGTCCTTTGGCGTGGTACTCTGGGAGATTGTGACCCTGGCAGAA

CAACCCTACCAGGGCCTGTCCAATGAGCAGGTGCTGAAGTTCGTCATGGATGGCGGGGTCCTGGAGGAGC

TGGAGGGCTGTCCCCTTCAGCTGCAGGAGCTGATGAGCCGCTGCTGGCAGCCGAACCCACGCCTGCGCCC

ATCTTTCACACACATTCTGGACAGCATACAGGAGGAGCTGCGGCCCTCCTTCCGCCTCCTCTCCTTCTAC

TACAGCCCGGAATGCCGGGGGGCCCGGGGCTCCCTGCCTACCACCGATGCAGAGCCTGACTCCTCACCCA

CTCCAAGAGACTGCAGCCCTCAAAATGGGGGTCCAGGGCACTGA

 

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.