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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.

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).
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.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes. Discard supernatant.
6. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels.

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