General Information for the Mouse ES Cell Resource

About the MicroRNAs (miRNA) Resource

MicroRNAs (miRNAs) are small non-coding RNA molecules known to play an important role in fundamental cellular processes through negative post-transcriptional regulation of gene expression. In an effort to address the role that microRNAs play in human cancer, their use as diagnostic tools, and their potential function as new targets for therapeutic intervention in the treatment of cancer, the Division of Cancer Biology (DCB) of the National Cancer Institute (NCI) has supported the generation of mouse embryonic stem cells (mESCs) harboring most known mouse miRNAs. In order to enhance novel information obtained on their role in cancer, 1501 genetically engineered mESC lines were produced, harboring conditional microRNA transgenes.

Drs. Gregory Hannon and Scott Lowe, at the Cold Spring Harbor Laboratory (CSHL), have utilized high-throughput technology to produce mES cells that conditionally express each known murine miRNA. The miR ESC lines, when used to generate genetically engineered mice, will produce a tet-inducible, reversible system to investigate the role of each miRNA in vivo, providing a powerful tool for the study of miRNA biology. Transgene expression for each ESC clone is controlled by either the tetracycline response element (TRE) or TRE-tight promoters, to accommodate a wide range of experimental designs. TRE-driven expression facilitates the study of microRNA in a variety of tissues, while TRE-tight promoter reduces leakiness and results in more restricted expression, suited for tissue-specificity, especially useful for study of miRNAs for which even the smallest level of expression may cause sterility or lethality (appendix 1). The following is the categorical distribution of the miR ESC lines:

  1. mES cell lines with mature microRNAs embedded in a miR30 precursor, designed to control the effect microRNA processing might have on overall expression
    a) 509 mESC lines expressing the miR controlled by TRE-tight promoter.
    b) 299 mESC lines expressing the major miR controlled by TRE promoter.
  2. mES cell lines with mature microRNA species in their endogenous context (referred to as: Primary-miRNAs)
    a) 261 mESC lines expressing the miR controlled by TRE-tight promoter.
    b) 432 mESC lines expressing the miR controlled by TRE promoter.

The parental ES cell line used to produce all the microRNA-expressing clones is KH2 (C57BL/6 x 129/Sv). It contains an frt-homing cassette and reverse tet-transactivator that were previously targeted into its ColA1 locus and Rosa26 loci (2). The miR clones were generated by electroporation of pColTtGM Flp-in targeting vector into the ES cells (appendix 2, see figure 3) containing either a TRE or a TRE-tight promoter and the fluorescent reporter gene GFP (green fluorescent protein). Presence of the tetracycline responsive transactivator M2-rtTA in the parental KH2 ES cells, allows for promoter-driven miR expression in the presence of doxycycline (dox). All ES cell lines generated with this inducible system have been validated by flow cytometry for GFP expression. Additionally, many have also been authenticated for miRNA expression as determined by RT-qPCR. These results are noted on the description page for each of the miRNA-containing mESC lines. Selection of validation methods was determined based on the following parameters:

  1. In some cases the endogenous expression of the miRs was not detectable in ES cells in the absence of dox assessed by Taqman miR qPCR, while their expression was observed in the presence of dox indicating their induction.
  2. In several cases, the expression levels of miRs were beyond saturated amounts that could be assessed by Taqman miR-qPCR, therefore the expression levels of their primary transcript were measured by RTqPCR, demonstrating the fold induction of the primary transcripts ranging from 4 to 55 for these miRs in miR-30 context.
  3. Induction of miR expression was unsuccessful in several clones even though the genomic sequences of the miRs for a select group among these were verified by PCR, possibly because the RNAi machinery could not efficiently process these mature miRs when placed into a miR-30 carrier.
  4. Validation for mESCs containing miRs larger than 22 bps was performed only by flow cytometry as miR-qPCR kits for these miRs were not readily available. Protocols utilized in the generation, care, manipulation, and use of the miR ESC clones are provided in the protocol section (appendix 3).

All miR ES cells from the collection are available for distribution through the NCI Mouse Repository. Requests for this resource may be placed through the this website. The requestor will be charged a nominal fee of $162.52 per ES cell line for processing and handling. Because the miR ES cell resource is limited, distribution to the scientific community* will be prioritized according to the following organizational affiliation:

*Available for domestic distribution only