Mouse PTCH/PTCH1/PTC/PTC1/Patched Lentivirus, Full-length Gene in Lentivector, Pre-packaged Lentiviral Particles

Description

PTCH/PTCH1/PTC/PTC1 is a member of the patched (PTCH) gene family. The PTCH gene encodes a 12-pass tranmembrrane protein containing 1 SSD (sterol-sensing) domain.  PTCH acts as a receptor for sonic hedgehog (SHH), a secreted molecule implicated in the formation of embryonic structures and in tumorigenesis, as well as indian hedgehog (IHH) and desert hedgehog (DHH). PTCH also associates with the smoothened (SMO) protien to transduce the hedgehog's proteins signal. PTCH may function as a tumor suppressor as inactivation of this gene is probably a necessary, if not sufficient step for tumorigenesis. Mutations of the PTCH gene are associated with basal cell nevus syndrome (BCNS), esophageal squamous cell carcinoma, trichoepitheliomas, transitional cell carcinomas of the bladder, as well as holoprosencephaly 7 (HPE7). Multiple transcript variants due to alternative splicing have been described but the full-length sequences and biological activity remain undetermined.

 

 

Gene Symbol: PTCH; PTCH1; PTC; BCNS; HPE7; PTC1; NBCCS; PTCH11

 

NCBI Gene ID: 19206

 

Uniprot Entry: Q61115

 

Construct Details: Full length mouse PTCH 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 Sequence:  

ATGGCCTCGGCTGGTAACGCCGCCGGGGCCCTGGGCAGGCAGGCCGGCGGCGGGAGGCGCAGACGGACCG

GGGGACCGCACCGCGCCGCGCCGGACCGGGACTATCTGCACCGGCCCAGCTACTGCGACGCCGCCTTCGC

TCTGGAGCAGATTTCCAAGGGGAAGGCTACTGGCCGGAAAGCGCCGCTGTGGCTGAGAGCGAAGTTTCAG

AGACTCTTATTTAAACTGGGTTGTTACATTCAAAAGAACTGCGGCAAGTTTTTGGTTGTGGGTCTCCTCA

TATTTGGGGCCTTCGCTGTGGGATTAAAGGCAGCTAATCTCGAGACCAACGTGGAGGAGCTGTGGGTGGA

AGTTGGTGGACGAGTGAGTCGAGAATTAAATTATACCCGTCAGAAGATAGGAGAAGAGGCTATGTTTAAT

CCTCAACTCATGATACAGACTCCAAAAGAAGAAGGCGCTAATGTTCTGACCACAGAGGCTCTCCTGCAAC

ACCTGGACTCAGCACTCCAGGCCAGTCGTGTGCACGTCTACATGTATAACAGGCAATGGAAGTTGGAACA

TTTGTGCTACAAATCAGGGGAACTTATCACGGAGACAGGTTACATGGATCAGATAATAGAATACCTTTAC

CCTTGCTTAATCATTACACCTTTGGACTGCTTCTGGGAAGGGGCAAAGCTACAGTCCGGGACAGCATACC

TCCTAGGTAAGCCTCCTTTACGGTGGACAAACTTTGACCCCTTGGAATTCCTAGAAGAGTTAAAGAAAAT

AAACTACCAAGTGGACAGCTGGGAGGAAATGCTGAATAAAGCCGAAGTTGGCCATGGGTACATGGACCGG

CCTTGCCTCAACCCAGCCGACCCAGATTGCCCTGCCACAGCCCCTAACAAAAATTCAACCAAACCTCTTG

ATGTGGCCCTTGTTTTGAATGGTGGATGTCAAGGTTTATCCAGGAAGTATATGCATTGGCAGGAGGAGTT

GATTGTGGGTGGTACCGTCAAGAATGCCACTGGAAAACTTGTCAGCGCTCACGCCCTGCAAACCATGTTC

CAGTTAATGACTCCCAAGCAAATGTATGAACACTTCAGGGGCTACGACTATGTCTCTCACATCAACTGGA

ATGAAGACAGGGCAGCCGCCATCCTGGAGGCCTGGCAGAGGACTTACGTGGAGGTGGTTCATCAAAGTGT

CGCCCCAAACTCCACTCAAAAGGTGCTTCCCTTCACAACCACGACCCTGGACGACATCCTAAAATCCTTC

TCTGATGTCAGTGTCATCCGAGTGGCCAGCGGCTACCTACTGATGCTTGCCTATGCCTGTTTAACCATGC

TGCGCTGGGACTGCTCCAAGTCCCAGGGTGCCGTGGGGCTGGCTGGCGTCCTGTTGGTTGCGCTGTCAGT

GGCTGCAGGATTGGGCCTCTGCTCCTTGATTGGCATTTCTTTTAATGCTGCGACAACTCAGGTTTTGCCG

TTTCTTGCTCTTGGTGTTGGTGTGGATGATGTCTTCCTCCTGGCCCATGCATTCAGTGAAACAGGACAGA

ATAAGAGGATTCCATTTGAGGACAGGACTGGGGAGTGCCTCAAGCGCACCGGAGCCAGCGTGGCCCTCAC

CTCCATCAGCAATGTCACCGCCTTCTTCATGGCCGCATTGATCCCTATCCCTGCCCTGCGAGCGTTCTCC

CTCCAGGCTGCTGTGGTGGTGGTATTCAATTTTGCTATGGTTCTGCTCATTTTTCCTGCAATTCTCAGCA

TGGATTTATACAGACGTGAGGACAGAAGATTGGATATTTTCTGCTGTTTCACAAGCCCCTGTGTCAGCAG

GGTGATTCAAGTTGAGCCACAGGCCTACACAGAGCCTCACAGTAACACCCGGTACAGCCCCCCACCCCCA

TACACCAGCCACAGCTTCGCCCACGAAACCCATATCACTATGCAGTCCACCGTTCAGCTCCGCACAGAGT

ATGACCCTCACACGCACGTGTACTACACCACCGCCGAGCCACGCTCTGAGATCTCTGTACAGCCTGTTAC

CGTCACCCAGGACAACCTCAGCTGTCAGAGTCCCGAGAGCACCAGCTCTACCAGGGACCTGCTCTCCCAG

TTCTCAGACTCCAGCCTCCACTGCCTCGAGCCCCCCTGCACCAAGTGGACACTCTCTTCGTTTGCAGAGA

AGCACTATGCTCCTTTCCTCCTGAAACCCAAAGCCAAGGTTGTGGTAATCCTTCTTTTCCTGGGCTTGCT

GGGGGTCAGCCTTTATGGGACCACCCGAGTGAGAGACGGGCTGGACCTCACGGACATTGTTCCCCGGGAA

ACCAGAGAATATGACTTCATAGCTGCCCAGTTCAAGTACTTCTCTTTCTACAACATGTATATAGTCACCC

AGAAAGCAGACTACCCGAATATCCAGCACCTACTTTACGACCTTCATAAGAGTTTCAGCAATGTGAAGTA

TGTCATGCTGGAGGAGAACAAGCAACTTCCCCAAATGTGGCTGCACTACTTTAGAGACTGGCTTCAAGGA

CTTCAGGATGCATTTGACAGTGACTGGGAAACTGGGAGGATCATGCCAAACAATTATAAAAATGGATCAG

ATGACGGGGTCCTCGCTTACAAACTCCTGGTGCAGACTGGCAGCCGAGACAAGCCCATCGACATTAGTCA

GTTGACTAAACAGCGTCTGGTAGACGCAGATGGCATCATTAATCCGAGCGCTTTCTACATCTACCTGACC

GCTTGGGTCAGCAACGACCCTGTAGCTTACGCTGCCTCCCAGGCCAACATCCGGCCTCACCGGCCGGAGT

GGGTCCATGACAAAGCCGACTACATGCCAGAGACCAGGCTGAGAATCCCAGCAGCAGAGCCCATCGAGTA

CGCTCAGTTCCCTTTCTACCTCAACGGCCTACGAGACACCTCAGACTTTGTGGAAGCCATAGAAAAAGTG

AGAGTCATCTGTAACAACTATACGAGCCTGGGACTGTCCAGCTACCCCAATGGCTACCCCTTCCTGTTCT

GGGAGCAATACATCAGCCTGCGCCACTGGCTGCTGCTATCCATCAGCGTGGTGCTGGCCTGCACGTTTCT

AGTGTGCGCAGTCTTCCTCCTGAACCCCTGGACGGCCGGGATCATTGTCATGGTCCTGGCTCTGATGACC

GTTGAGCTCTTTGGCATGATGGGCCTCATTGGGATCAAGCTGAGTGCTGTGCCTGTGGTCATCCTGATTG

CATCTGTTGGCATCGGAGTGGAGTTCACCGTCCACGTGGCTTTGGCCTTTCTGACAGCCATTGGGGACAA

GAACCACAGGGCTATGCTCGCTCTGGAGCACATGTTTGCTCCCGTTCTGGACGGTGCTGTGTCCACTCTG

CTGGGTGTACTGATGCTTGCAGGGTCCGAATTTGATTTCATTGTCAGATACTTCTTTGCCGTCCTGGCCA

TTCTCACCGTCTTGGGGGTTCTCAATGGACTGGTTCTGCTGCCTGTCCTCTTATCCTTCTTTGGACCGTG

TCCTGAGGTGTCTCCAGCCAATGGCCTAAACCGACTGCCCACTCCTTCGCCTGAGCCGCCTCCAAGTGTC

GTCCGGTTTGCCGTGCCTCCTGGTCACACGAACAATGGGTCTGATTCCTCCGACTCGGAGTACAGCTCTC

AGACCACGGTGTCTGGCATCAGTGAGGAGCTCAGGCAATACGAAGCACAGCAGGGTGCCGGAGGCCCTGC

CCACCAAGTGATTGTGGAAGCCACAGAAAACCCTGTCTTTGCCCGGTCCACTGTGGTCCATCCGGACTCC

AGACATCAGCCTCCCTTGACCCCTCGGCAACAGCCCCACCTGGACTCTGGCTCCTTGTCCCCTGGACGGC

AAGGCCAGCAGCCTCGAAGGGATCCCCCTAGAGAAGGCTTGCGGCCACCCCCCTACAGACCGCGCAGAGA

CGCTTTTGAAATTTCTACTGAAGGGCATTCTGGCCCTAGCAATAGGGACCGCTCAGGGCCCCGTGGGGCC

CGTTCTCACAACCCTCGGAACCCAACGTCCACCGCCATGGGCAGCTCTGTGCCCAGCTACTGCCAGCCCA

TCACCACTGTGACGGCTTCTGCTTCGGTGACTGTTGCTGTGCATCCCCCGCCTGGACCTGGGCGCAACCC

CCGAGGGGGGCCCTGTCCAGGCTATGAGAGCTACCCTGAGACTGATCACGGGGTATTTGAGGATCCTCAT

GTGCCTTTTCATGTCAGGTGTGAGAGGAGGGACTCAAAGGTGGAGGTCATAGAGCTACAGGACGTGGAAT

GTGAGGAGAGGCCGTGGGGGAGCAGCTCCAACTGA

 

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.