Rene H. Quintanilla1, Andrew Fontes1, Jennifer C. Moore2, Ronald P. Hart2, Uma Lakshmipathy1

1Primary and Stem Cell Systems, Life Technologies, 5781 Van Allen Way, Carlsbad, CA2Rutgers Stem Cell Research Center, Rutgers University, 604 Allison Road, Piscataway, NJ 08854

RESULTS

Figure 1. BacMam Platform

Figure 4: DGCR8 RNAi expression in AdSC inhibits Adipogenesis

Adipose derived stem cells were transduced with BacMam CMV-GFP control and BacMam DGCR8 RNAi were subjected to 7 days of adipogenesis. Cells were fixed and labeled with Fatty Acid Binding Protein-4 (green) and LipiTOX Red to demonstrate emerging adipocytes. Control cells demonstrate a high number of lipid deposits (phase) and adipocyte markers, while cell treated with the BacMam RNAi demonstrates a decrease in the number of cells that underwent differentiation .

CONCLUSIONS o BacMam allows expression tracking via co-cistronic expression of GFP along with the RNAi

construct expression. This allows the efficient monitoring in downstream applications. This can potentially allow multiple gene knockdowns..

o DGCR8 knockdown allows large scale validation of unique cell specific miRNAs. DGCR8 RNAi BacMam containing as polycistronic GFP cassette allowed for transduction and visualization of the RNAi transduced cells.

o DGCR8 RNAi treated cells showed robust expression of GFP and decreased levels of DGCR8 proteins as assessed by Western blot analysis.

o DGCR8 gene knockdown in AdSC shows decreased adipogenesis with no visible impact on osteogenesis.

o Further Large scale genome and epigenome analysis is underway to tie the cell effect to gene/epigenome changes resulting from DGCR8 Knock down.

REFERENCESRP Hart, J Davila, and U Lakshmipathy MicroRNA in Pluripotent stem cells, A review. Regenerative Medicine. Vol 5(4), p545, 2010U Lakshmipathy and RP Hart (2008) Micro RNA expression in MSC: A Concise Review. Stem Cell . 26, 356-363.LA Goff, S Boucher, C Ricupero, S Fenstermacher, M Swerdel, L Chase, C Adams, JD Chesnut, U.Lakshmipathy, RP Hart (2008) Differentiating human MSC regulate miRNAs: Prediction of microRNA regulation by PDGF during osteogenesis. Experimental Hematology, 36, 1354-1369.U Lakshmipathy, B Love, LA Goff, R Jornsten, R Graichen, RP Hart, JD Chesnut (2007) MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells. Stem Cells and Development 16, 1003-1016.R Josephson, CJ Ording, Y Liu, S Shin, U Lakshmipathy, A Toumadje, B Love, JD Chesnut, PW Andrews, MS Rao, JM Auerbach (2007) Qualification of embryonal carcinoma 2102Ep as a reference for human embryonic stem cell research Stem Cells 25, 437-446.

ACKNOWLEDGEMENTS We thank Kelly Scheyhing for initial help with insect cell culture; Andrew Fontes and David Thompson for technical help with cloning; and Drs Bhaskar Thyagarajan, Mohan Vemuri and Jon Chesnut for their suggestions and discussions. This work was supported by Primary and Stem Cell Systems, Life Technologies.

Development of Stem Cell based Assays using BacMam platform.

pBacMamTM2-DEST-CMV is a second generation (Gen2) vector with additional VSV-g coat protein and WPRE element for enhanced delivery and robust expression (A). The construct comprises attR1 and attR2 sites that can be utilized for rapid cloning using MuttiSite Gateway cloning (B). Cloning into BacMam of RNAi contructs for knockdown of DGCR8 along with a GFP reporter in multicistronic cassettes.(C). BacMam platform is suitable for diverse expression systems for downstream assays

Figure 5:DGCR8 expression in AdSC has no effect on osteogenesis

B

Figure 3: DGCR8 RNAi 1-1 BacMam expression and effect on endogenous DGCR8 protein levels.

AB

ABSTRACTEmerging epigenetic analysis of stem cells demonstrates the integral role of microRNAs as regulators for pluripotency maintenance, proliferation and differentiation. Novel genome-wide analysis tools have enabled identification of 500-1000 novel species of microRNAs that may have regulatory roles in pluripotency. Here, we report the development of a cell-based validation tool to confirm and elucidate the cellular functions of these novel microRNAs. Utilizing the BacMam platform we have developed an efficient method for RNAi delivery to functionally knock-down DGCR8, a key component in the microRNA biosynthesis pathway. DGCR8 knockdown stem cells thusly generated will allow experimental confirmation that the novel microRNAs, predicted from SOLiD datasets, are produced by the traditional Microprocessor pathway, essentially confirming their identities as microRNAs. The DGCR8 RNAi BacMam with a GFP cassette for visualization of transduced cells was tested in Ntera2 human embryonal carcinoma cells and in adipose derived mesenchymal stem cells. Preliminary results indicate a decrease in endogenous levels of the DGCR8 level with RNAi expression and the induction of distinct cellular phenotypes. Further genome wide evaluation of gene and epigenetic expression will allow confirmation of large numbers of miRNAs and will establish a system for testing their potential role in stem cell maintenance and differentiation.

C

Multiple DGCR8 expressing cell lines transduced with BacMam DGCR8 RNAi show persistence in GFP expression (A) .Western blot analysis of DGCR8 protein expression decreased levels of DGCR8 levels in multiple cells types (B).

GFP

5` miR

3` miRPre-miRNA/ShRNA

AdSC GFP Control DGCR8 RNAi

FABP4 FABP4FABP4LipiTOX Red LipiTOX RedLipiTOX Red

INTRODUCTIONMicroRNA biogenesis involves a microprocessor complex that is required to process primary miRNAs transcripts (pri-miRNAs) to release precursor miRNAs (pre-miRNAs) in the nucleus. A key component of the microprocessor complex, DGCR8 (DiGeorge Syndrome Critical Region 8) acts in a feedback loop which has been implicated in ESC cell cycle control and differentiation potential. Currently there are few tools for validation of a large number of these known and novel microRNAs. RNAi expression systems can be used to functionally knock-down DGCR8 to validate these miRNAs and elucidate their the complex interactions and pathways.

BacMam has been utilized for efficiently delivery genes into several hard-to-transfect cell types such as mesenchymal stem cells and their derivatives, hepatic cell model systems and several primary neuronal subtypes. Here utilize we use our BacMam platform for efficient delivery of RNAi constructs in a transient, foot- print free method. .

Figure 2. Evaluation of DGCR8 RNAi BacMam in NTera2 human embryonal carcinomal cells

(A) High Expression of DGCR8 RNAi as measured via expression of GFP in transduced Ntera cells. (B) Western blot analysis of cells treated without and with DGCR8 RNAi shows a band at ~80-85kB that is expressed in untransduced and GFP control cells but relatively decreased to varying levels in the four different oligos. Oligo1 was used for further studies.

A

B

A

Scheme 1: Role of various Microprocessor enzymes in the biogenesis of mature miRNA .

Cells harvest at 48h post transduction and seeded at appropriate density to initiate osteogenesis showed no significant difference in morphology or cells between the untransduced and GFP BacMam transduced controls with the DGCR8 RNAi transduced cells. Consistent with this observation, no obvious difference was noted in ELF97 alkaline phophatase staining pattern of the differentiating osteoblasts between the three conditions.