Dicer1-/- (ATCC® CRL-3221)

Organism: Mus musculus, mouse  /  Cell Type: mesenchymal stem cell  /  Tissue: Derived from: mesenchyme  / 

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Organism Mus musculus, mouse
Tissue Derived from: mesenchyme
Cell Type mesenchymal stem cell
Product Format frozen 1.0 mL
Morphology mesenchymal-like
Culture Properties adherent
Biosafety Level 2  [Cells contain SV40 viral DNA sequences]

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Age 13 month
Gender female
Applications Dicer1-/- mesenchymal stem cells (ATCC® CRL-3221™) lack expression of the Dicer1 protein. Dicer1-/- cells can be used in conjunction with the matching Dicer1f/f cells (ATCC® CRL-3220™) to study the role of Dicer and miRNA in cell biology.
Storage Conditions liquid nitrogen vapor phase
Images Cell Micrograph of Dicer1-/- ATCC CRL-3221
Derivation The cells were first derived as primary cultures from a Dicer1f/f mouse with loxP sites present on either side of exon 23 of the Dicer1 gene. The primary culture was then infected with ecotropic virus encoding SV40 large T-Antigen, and immortalized. The immortalized MSCs then were infected with Adeno-Cre-GFP to introduce Dicer gene knockout by Cre/loxP- mediated recombination. The cells were sorted for GFP. Clones were then isolated and genotyped.
Genes Expressed functional Dicer1 not expressed
Comments

MicroRNAs (miRNAs) are a new class of small noncoding RNAs that post-transcriptionally regulate the expression of target mRNA transcripts. Dicer is an RNase III family endoribonuclease that has the biofunction of processing miRNAs. Loss of Dicer1 leads to a dramatic decrease in levels of cellular microRNA, allowing for investigations into the roles of microRNAs in various cellular functions.

CRL-3221 the Dicer1-/- cell line is a model of homozygous Dicer1-deletion in murine mesenchymal stem cells established from an adult Dicer1f/f mouse and immortalized in vitro. CRL-3220 is the Dicer1f/f immortalized mouse mesenchymal stem cell line that can be paired with CRL-3221 and used as a control.

Complete Growth Medium Alpha minimum essential medium with ribonucleosides and deoxyribonucleosides, 90%; fetal bovine serum, 10%
Subculturing Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin - 0.53mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
  3. Add 1.0 to 2.0 mL of 0.25% Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
  4. Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37.0°C to facilitate dispersal.
  5. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  6. Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes. Discard supernatant.
  7. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:5 is recommended
Medium Renewal: 2 to 3 times per week
Seeding Density: 1.5 x 104 to 3.0 x 104 cells/cm2
Cryopreservation Freeze Medium: Fetal Bovine Serum (FBS), 92%; DMSO, 8%
Storage Temperature: liquid nitrogen vapor phase
Culture Conditions Temperature:37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Volume 1.0 mL
Name of Depositor A Gurtan (P Sharp's lab)
Year of Origin 2009
References

Ravi A, et al. Proliferation and tumorigenesis of a murine sarcoma cell line in the absence of DICER1. Cancer Cell 21(6): 848-855, 2012. PubMed: 22698408

Gurtan AM, et al. In vivo structure-function analysis of human Dicer reveals directional processing of precursor miRNAs. RNA 18: 1116-1122, 2012. PubMed: 22546613

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • For-profit customers must obtain a research use license agreement from the Contributor prior to shipment.
  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
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Restrictions

This material is subject to the following restrictions in addition to those outlined in the ATCC Material Transfer Agreement:

  1. Prior to purchase, any for-profit Purchaser must obtain a research use license from Massachusetts Institute of Technology; and
  2. When a Commercial Use is contemplated, all for-profit and non-profit Purchasers must obtain a Commercial Use license from Massachusetts Institute of Technology.  Please contact Massachusetts Institute of Technology at the address listed below:
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Technology Licensing Office, NE18-501
Cambridge, Massachusetts 02142
617-253-6966 tlo@mit.edu

References

Ravi A, et al. Proliferation and tumorigenesis of a murine sarcoma cell line in the absence of DICER1. Cancer Cell 21(6): 848-855, 2012. PubMed: 22698408

Gurtan AM, et al. In vivo structure-function analysis of human Dicer reveals directional processing of precursor miRNAs. RNA 18: 1116-1122, 2012. PubMed: 22546613