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| Status |
Public on Oct 02, 2012 |
| Title |
Gene Expression Changes in a Tumor Xenograft by a Py-Im Polyamide |
| Organisms |
Homo sapiens; Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Gene regulation by DNA binding small molecules could have important therapeutic applications. This study reports the investigation of a DNA-binding pyrrole-imidazole polyamide targeted to bind the DNA sequence 5’-WGGWWW-3’ with reference to its potency in a subcutaneous xenograft tumor model. The molecule is capable of trafficking to the tumor site following subcutaneous injection and modulates transcription of select genes in vivo. A FITC-labeled analogue of this polyamide can be detected in tumor-derived cells by confocal microscopy. RNA deep sequencing (RNA-seq) of tumor tissue allowed the identification of further affected genes, a representative panel of which were interrogated by qRT-PCR and correlated with cell culture expression levels.
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| Overall design |
Xenografts. Grafting with A549-luc-C8. Experiments were performed in female SCID-beige mice (Charles River) between 8 and 12 weeks of age. Cells were injected into the left flank area of the animals as suspensions of 25 x 106 mL-1 in RPMI, 200 µL per injection. Treatment and tumor proliferation monitoring. Mice were treated following the schedule delineated in treatment protocol. Tumor proliferation was monitored using the XENOGEN imaging device. The animals were anesthetized with 2 5 % isoflurane and subsequently transferred to the imaging chamber, whereupon the isoflurane levels were reduced to 1-2.5 %. The floor of the imager was heated to +37 ºC to avoid hypothermia. Breathing frequency was monitored and not allowed to drop below 1 s-1, adjusting the isoflurane levels accordingly at all times. Endpoint criteria and euthanasia. Animal endpoint criteria encompassed weight loss of over 15 %, restriction of motor function by the engrafted tumor, dehydration of over 10 % and moribund behavior. Where appropriate, the animals were euthanized by asphyxiation in a CO2 chamber. Tumor tissue harvest. Animals were resected and tumors excised using standard forceps, scissors and surgical blades. The tumors were combined into one sample per condition and mechanically sheared in TRIZOL, employing a specialized device (tissue tearer, model 985370). Total RNA workup was performed following the standard TRIZOL procedure, followed by a DNAse digest.
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| Contributor(s) |
Raskatov JA, Nickols NG, Hargrove AE, Marinov GK, Wold B, Dervan PB |
| Citation(s) |
22988074 |
| |
| Submission date |
Aug 20, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Jevgenij A. Raskatov |
| E-mail(s) |
raskatov@caltech.edu
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| Phone |
6263956084
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| Organization name |
CalTech
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| Department |
CCE
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| Lab |
Dervan
|
| Street address |
1200 EAST CALIFORNIA BOULEVARD
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| City |
PASADENA |
| State/province |
California |
| ZIP/Postal code |
91125 |
| Country |
USA |
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| Platforms (2) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
| GPL13112 |
Illumina HiSeq 2000 (Mus musculus) |
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| Samples (10)
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| Relations |
| BioProject |
PRJNA173215 |
| SRA |
SRP014920 |
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