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Can the double-fluorescent reporter mouse (ROSAnT/nG) be used to improve uterine smooth muscle (USM) specific protein knockdown in murine parturition studies.
Abstract Number: SAT-14
Abstract Type: Original Research
Objective: Assessing the necessary molecular targets in the initiation and maintenance of labor is an important goal to develop effective and novel approaches to treating pre-term labor. Currently, inducible gene-editing approaches (ie. Cre/Flox systems) provide the most reliable method for highly selective protein targeting. However, these approaches can be limited due to differential tissue expression and variability in induction. Furthermore, only a few reports exist using these systems to knockdown USM proteins involved in parturition and none provide a means for selection of successfully targeted USM cells. Novel double-fluorescent reporter mice (ROSAnT/nG) provide a unique means to assess tissue knockdown by changes in fluorescence (red changes to green). We questioned if ROSAnT/nG/Cre ERT2 recombination could be a useful tool to precisely target protein knockdown in murine USM tissue and cells.
Methods: With IACUC approval, we have begun triple breeding strategy to generate 2 mouse groups: ROSAnT/nG/ SM22wt (baseline control – Tamoxifen unresponsive); SM22-CreERT2/ROSAnT/nG (fluorescent reporter, tamoxifen responsive). All groups will receive tamoxifen (1 mg /100 µL) i.p. for 5 days and smooth muscle (uterus and small intestine) will be harvested 7 days after the last injection. Changes in fluorescent expression will be assessed in histological samples by confocal microscopy and quantified by FACS cell sorting.
Results: Preliminary results show high RFP expression in uterine smooth muscle in the ROSAnT/nG baseline mice that does not alter under tamoxifen treatment (Figure 1).
Conclusions: Control studies reveal excellent RYP expression in our baseline mouse. Tamoxifen treatment (in our treatment control group) did not convert RFP into GFP – consistent with our hypothesis. We are awaiting sufficient female SM22-CreERT2/ROSAnT/nG mice to assess the efficacy of this fluorescent reporting system.