Appendix 1

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Comparison of tissue specificity of GFP expression in TRE-tight- let-7c and TRE-let-7c genetically engineered mice

A comparison of the expression pattern of GFP in various organs from TRE-tight-versus TRE-let-7c mice is provided (see Figure 1). Consistent with previous reports (1, 3), significant variation in the level of GFP induction between different organs from TRE-mice (heterozygous let-7c/heterozygous rtTA2) was observed. Imaging analysis showed that GFP fluorescence was readily detected in the thymus, skin, small intestine, and colon (not shown) from TRE-let-7c as well as TRE-tight-let-7c when doxycycline (dox) in drinking water was given to mice for four days. However, GFP positivity was only observed in lung, kidney and liver from TRE-let-7c mice with the presence of a single R26-rtTA transgenic allele (labeled as hetero-rtTA2; see #2 organs in Figure 1), but not from TRE-tight-let-7c mice (see #3 organs in Figure 1). Previously, the presence of two R26-rtTA transgenic alleles increased the dox-dependent expression of GFP-shRNAs relative to a single allele in TRE-mice was reported (1). However, no GFP positivity was observed in kidney, lung and liver from TRE-tight-let-7c mice even in the presence of two R26-rtTA alleles (labeled as homo-rtTA; see #4 organs in Figure 1).

Finally, rtTA-dependent GFP induction in spleen from TRE-tight let-7c mice was observed (Figure 1). The level of GFP fluorescence with two R26-rtTA alleles in TRE-tight-let-7c mice was comparable to the level with one R26-rtTA allele in TRE-let-7c mice (Figure 1, #2 vs. #4 organs). These results suggested that TRE-tight promoter-driven GFP induction was more tissue-restricted than TRE promoter-driven GFP induction. In homozygous ROSA mouse strains with other shRNAs driven by TRE-tight promoter, fluorescence was observed in more widespread tissues including the pancreas, testis, and uterus, which could be comparable to the levels seen in mice having heterozygous ROSA with TRE promoter (data not shown). In the bone marrow, 60-80 % cells were GFP positive, though fluorescence in the liver, kidney, lung, and heart from TRE-tight was below the sensitivity threshold of the imaging assay. Speckled GFP positive cells were detected by immunohistochemistry in the lung, kidney and liver (data not shown). The increased expression of let-7c in the intestine, spleen and thymus from TRE-tight-let-7c mice was confirmed using small RNA northern analysis. In contrast to the restricted tissue distribution of GFP fluorescence in adult tissues, much stronger GFP induction was observed in most of the tissues including liver and brain in embryos at E15.5 after dox was given to the mother for 4 days.

Comparison of GFP expression in varios orgains from TRE vs TREtight mice Comparison of GFP expression in varios orgains from TRE vs TREtight mice

Appendix 2

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MicroRNA ES cell resource construction

The miRNA ES cell resource was generated in the following four phases, carried out concurrently, to achieve the goal of generating all 1501 miRNA-harboring mESC:

1) Construction of the recipient pColTtGM Flp-in targeting vectors:
The frt-mediated targeting strategy was applied to generate ES cells expressing regulatable miRNA. KH2 ES cells were used in which an frt-homing cassette and reverse tet-transactivator were previously targeted into ColA1 locus and Rosa26 loci (2). Expression of FLPe Recombinase mediates recombination between the frt site on pColTGM vector and those within the homing cassette at the ColA1 locus (see figure 3).

It has been shown that miR30-based shRNAs can be inducibly expressed in a tissue specific and reversible manner (3). The miR30 cassette serves as the backbone to receive predicted 22nt shRNA, included in the Hannon-Elledge genome library. Therefore, the pBS31 vector was modified by the insertion of miR-30 based cassette downstream of GFP to generate the recipient targeting vector (called pColTtGM, see Figure1A). Additionally, the TRE promoter was replaced with a TRE-tight version to reduce leaky expression of either shRNAs/miRNAs. The miR-30 based cassette contains convenient and unique XhoI and EcoRI sites for rapid single-step cloning of miRNA expression targeting vectors to generate the library. Moreover, for pri-miRNA expression, the miR30 cassette was replaced with a linker, having multiple cloning sites, to generate the new targeting vector (called pColTtG-linker, see figure 1B) for construction of the library of targeting vectors expressing pri-miRs. Sequences for all recipient vectors were verified using 5 different sets of sequencing primers.

Figure 1. Schematic diagram of recipient Flp-in targeting vectors. A) CTtGM as a recipient vector to construct Flp-in targeting vectors carrying miRs in a miR30 carrier. B) CTtG-linker recipient vector to construct Flp-in targeting vectors harboring Pri-miRNAs.

Figure 1. Schematic diagram of recipient Flp-in targeting vectors.
A) CTtGM as a recipient vector to construct Flp-in targeting vectors carrying miRs in a miR30 carrier. B) CTtG-linker recipient vector to construct Flp-in targeting vectors harboring Pri-miRNAs.

2) Design the libraries of oligonucleotides of mature miRNAs (embedded in miR30 precursor) and the library of individual pri-miRs:

CSHL has generated populations of 96-mers of 680 miR-30 based mature miRNAs which contain exact complementary to the known mature miRNA sequences on a surface chip (Agilant Technology,CA). These oligonucleotides were used as templates to produce PCR products for the construction of the libraries of miRNA-express-targeting vectors. Sequences of PCR primers for the amplification of the library of oligonucleotides follow:

a) miRXho-F; 5' -tacaatactcgagaaggtatattgctgttgacagtgagcg-3'
b) miREco-R; 5' -acttagaagaattccgaggcagtaggca-3

In addition, two sets of 487 PCR primers were designed by Open Biosystems for the amplification of pri-miRNA transcripts. Each primer contains a combination of XhoI, SalI, or BclI and EcoRI or MfeI or MluI in the 5' and 3' end respectively. The mouse genomic DNA was used as a template for PCR to amplify pri-miRs. Predicted 431 pri-miRNA-PCR products were amplified and resolved on an agarose gel, stained with ethidium bromide and photographed (See Figure 2). The mouse genomic DNA was used as a template for PCR, for the amplification of pri-miRs. Predicted pri-miRNA- PCR products, ranging from 200 bps to 1500 bps, were resolved on the agarose gel, stained with ethidium bromide, and photographed (See Figure 2).

Figure 2. The representative photographic image of 98 PCR-products of primary miRNAs ampliflied using mouse genomic DNA and primers correspoding to the 5' and 3' ends of predicted pri-miRNA sequences.

Figure 2. The representative photographic image of 98 PCR-products of primary miRNAs ampliflied using mouse genomic DNA and primers correspoding to the 5' and 3' ends of predicted pri-miRNA sequences.

3) Production of distinct libraries of pColTtG targeting vectors harboring miRNAs:

a. Production of the library of targeting vectors carrying miR-30-based miRNAs

The PCR products (110 bps) of miR-30-based miRNAs were amplified using oligonucleotides from Agilant chips, digested with XhoI and EcoRI, and ligated into the same sites within pColTtGM vector. Correct clones were identified by sample sequencing of the pool. Forty five hundred colonies on the plates were sequence analyzed at the Cold Spring Harbor Lab Sequencing Facility. Sequencing results showed that 375 miRNAs out of a total of 680 miRNAs (55%) were successfully cloned into targeting vector, pColTtGM. The miniprep-DNAs were stored in 96-well plates. Individual targeting vectors would be re-prepped by Qiagen Maxi prep (Qiagen, CA) and sequence verified again prior to transfection into ES cells.

b. Production of library of targeting vectors expressing Pri-miRs

Due to various sizes and different availability of cloning sites of each PCR products, PCR products were pooled by three sets of sizes (200-500, 500-1000, and 100-1500 bps) and four sets of cloning sites (Xho or BclI as a 5 site, and EcoRI or MluI as a 3' site).

4) Production of ES cell lines harboring miRNAs and validation by flow cytometry and RT-qPCR:

Established FRT mediated gene targeting protocols were used (2) with modifications (figure 3). Briefly described, FLPe recombinase (2.5 μg) and Flp-in targeting vectors (5 μg) were transfected into KH2 cells using a nucleofector 96-well shuttle system (Lonza, Germany). Expression of FLPe mediates recombination between frt sites at the ColA1 locus in KH2 cells and those in the targeting vector. Since correct integration of targeting vector at ColA1 locus confers hyromycin resistance and neomycin sensitivity, positive clones were selected with hygromycin (140 μg/ml). Four different single clones per miRNAs were picked about 11-13 days post-electroporation. Correct single-site integration was confirmed by Southern Blotting (results not shown). Cells were then plated into 24-well plates and grown to confluence. They were then expanded to 6-cm plates and used to make frozen stocks. A subpopulation of these cells was re-plated onto a 24-well plate and used for flow cytometric analysis and RNA purification. Since KH2 cells express rtTA (reverse tet-transactivator), transgene expression can be induced in the ES cells by addition of doxycycline and assessed by flow cytometric analysis and RT-qPCR respectively. Forty-eight hours post-doxycycline treatment, cells were either lysed into Trizol solution for RNA purification, or trypsinized to measure GFP levels using Guava flow cytometer (Millipore, CA). RT-qPCRs were performed using Taqman-microRNA assay kit (Applied Biosystems, ID # 001141).

Figure 3. (a) Strategy for Flpe-mediated recombination at the <em>ColA1</em> locus in KH2 mES cells. Previously engineered KH2 cells contain, at independent loci, <em>Rosa26</em>-M2rtTA and the 'FRT homing cassette' consisting of <em>PGK</em>-Neo<sup>R</sup> and a nonfunctional hygromycin resistance cassette with no ATG site or promoter Co-electroporation of the ColFLp-TGM and pCAGGs-Flpe Recombinase vectors promotes inter- and intra-chromosomal recombination at the <em>ColA1</em> locus downstream of the Type I Collagen gene, resulting in excision of <em>PGK</em>-Neo<sup>R</sup> and integration of the ColFLp-TGM. Correct integration restores hygromycin resistance.
Figure 3. (a) Strategy for Flpe-mediated recombination at the ColA1 locus in KH2 mES cells. Previously engineered KH2 cells contain, at independent loci, Rosa26-M2rtTA and the "FRT homing cassette" consisting of PGK-NeoR and a nonfunctional hygromycin resistance cassette with no ATG site or promoter Co-electroporation of the ColFLp-TGM and pCAGGs-Flpe Recombinase vectors promotes inter- and intra-chromosomal recombination at the ColA1 locus downstream of the Type I Collagen gene, resulting in excision of PGK-NeoR and integration of the ColFLp-TGM. Correct integration restores hygromycin resistance.

Appendix 3

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ES Cell Culture and Maintenance

The following list of supplies and reagents are needed:

Item Vendor Catalog No.
KNOCKOUT DMEM Invitrogen 10829-018
0.25% Trypsin-EDTA Invitrogen 25200-056
β-mercaptoethanol Sigma M7154
FBS (ES cell qualified) Stem Cell Technologies 6952
Gelatin Millipore G-2500
L-Glutamine, 200mM Invitrogen 25030-081
LIF(ESGRO) Millipore ESG1107
Penicillin G ICN 194537
Streptomycin sulfate Invitrogen 11860-038
D-PBS Invitrogen 10010-023

Media Preparation

M15 Growth Media

GPS Solution (Glutamine/Penicillin/Streptomycin)

100 X ß-mercaptoethanol (BME)

MEF Feeder Media

Freezing Media (10% DMSO)

ES Cell Maintenance and Culture

Preparation of feeder plates

  • Add the appropriate volume of 0.1% gelatin solution to the plates and leave at room temperature for 30–60 minutes (See below: Table 1)
  • Aspirate the gelatin solution
  • Immediately add the appropriate volume and concentration of mitotically inactive MEF feeder cells (treated with mitomycin C or irradiated) to the plates (See below: Table 1)
  • Size of TC Plate Volume of Gelatin No. of Feeder Cells per Plate Volume of Media per Plate
    24-well 0.5 ml 1x10^5
    6-well 1.5 ml 5x10^5
    60 mm 2.0 ml 7x10^5
    100 mm 3.0 ml 1x10^6

Thawing the vial

Each vial contains a minimum of 2x106 ES cells and may be thawed on a 60 mm feeder plate:

Note: ES cells should be passaged every 2–3 days, and media should be changed prior to turning yellow.

Passaging ES cells

ES cells may be split 1:3 to 1:10:

Preparing ES cells for microinjection

ES cell culture after 24 hours, at approximately 40% confluence

Note: ES cell colonies with bright edges and a smooth, three-dimensional appearance.

ES cell colonies with bright edges and smooth three-dimensional appearance

Freezing ES cells

Appendix 4

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References

  1. Premsriut, P.K., Dow, L. E., Kim, S. Y., Camiolo, M., Malone, C.D., Miething, C., Scuoppo, C., Zuber, J., Dickins, R.A., Kogan, S., Shroyer, K.R., Sordella, R., Hannon, G.J., Lowe, S.W. (2011), A rapid and scalable system for studying gene function in mice using conditional RNA interference. Cell. 145(1):145-58
  2. Xue, W., Zender, L., Miething, C., Dickins, R.A., Hernando, E., Krizhanovsky, V., Cordon-Cardo, C., Lowe, S.W. (2007). Senescence and tumour clearance is triggered by 53 restoration in murine liver carcinomas. Nature. 445:656-60.
  3. Beard, C., Hochedlinger, K., Plath, K., Wutz, A., Jaenisch, R. (2006). Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis. 44: 23-28.
  4. Reinhart, B.J, Slack, F.J., Basson, M, Pasquinelli, A.E,, Bettinger, J.C., Rougvie, A.E., Horvitz, R., Ruvkun, G. (2000). The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature. 403: 901-6.
  5. Boyerinas, B., Park, S.M., Hau, A., Murmann, A.E., Peter, M.E. (2010) The role of let-7 in cell differentiation and cancer. Endo Relar Cancer. 29: 19-36.