This work was supported in part by the Research Scholar Grants (116403-RSG-09-082-01-MGO) from the American Cancer Society and intramural funds of Wake Forest University Health Sciences to GS. DBS was supported by NCI training grant 5T32CA079448.

1. Generation of shRNA Constructs
<ol>
	<li>Identify a siRNA-target sequence (20-23 nucleotides) based on previously published criteria6 or use a web-based algorithm server, such as "siRNA Target Finder" from Ambion and GenScript.</li>
	<li>Synthesize oligonucleotides based on the design of Figure 1 and example sequences in Table 1. Carry out PCR amplification using the oligonucleotides P1 and P2 (30 cycles of denaturing at 94 &deg;C for 30 sec, annealing at 60 &deg;C for 30 sec, and elongating at 72 &deg;C for 30 sec) and a plasmid carrying the U6 or H1 promoter as a template. Purify the PCR product using a PCR purification column. Digest it by BamHI and HindIII and purify the digested DNA using a PCR purification column. Anneal the oligonucleotides P3 and P4 (2.5 pmol/&mu;l of each in 100 &mu;l of 50 mM Tris&bull;HCl, pH 8.0) by boiling for 5 min and slowly cool down to ambient temperature. Digest a lentiviral vector, such as the one described in Figure 2A, by BamHI and EcoRI, and perform gel-purification.</li>
	<li>Carry out a 3-fragment ligation with the BamHI/EcoRI-digested vector, a BamHI/HindIII-digested PCR fragment and one pair of annealed oligonucleotides (forming HindIII and EcoRI sites at the two ends) at a 1:10:10 molar ratio (Figure 1).</li>
	<li>Transform highly efficient competent E. coli DH5&alpha; cells using the ligated product. Screen the colonies using the oligonucleotides P5 (in the vector) and P6 (in the H1 or U6 promoter).7</li>
	<li>Prepare plasmid DNA from the positive colonies with a commercial kit and confirm the presence of the insert by BamHI/EcoRI digestion and DNA agarose electrophoresis. Sequence the region containing the promoter and shRNA using oligonucleotide P5.</li>
	<li>Use plasmid DNA Midi- or Maxi-preparation kits (endotoxin-free is preferred) to prepare the lentivirus DNA carrying the shRNA expression cassette. Determine the DNA concentration by measuring the absorption at 260 nm. Check the DNA purity using the 260 nm/280 nm ratio and make sure it falls in the range of 1.8 to 2.0. Check the lentivirus plasmid integrity using agarose gel electrophoresis. DNA for transfection should be super-coiled.</li>
</ol>
2. Lentivirus Transfection
Precautions: Lentiviral vectors are derived from the human immunodeficiency virus-1 (HIV-1) genome and replication incompetent. They have been widely used in gene delivery due to the capability of infecting both dividing and non-dividing cells. However, two major risks exist in the studies using lentivirus. (1) The potential for generation of replication-competent lentivirus. This risk can be greatly reduced if the third generation lentiviral system is used. (2) The potential for oncogenesis. This risk can be exacerbated if the carried inserts are oncogenic or repress tumor suppressors. Research activities involved in HIV-based lentivirus should follow the "Biosafety Considerations for Research with Lentiviral Vectors" of the NIH (<a href="http://oba.od.nih.gov/rdna_rac/rac_guidance_lentivirus.html" target="_blank">http://oba.od.nih.gov/rdna_rac/rac_guidance_lentivirus.html</a>) and require an approval from the Biosafety Committee of a researcher's institution. Generally, enhanced Biosafety level-2 (BSL-2) containment is required for the laboratory setting if lentiviral vectors are used.
<ol>
	<li>Plate 4 &times; 106 HEK 293T cells with complete DMEM medium (DMEM supplied with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, 100 &mu;g/ml streptomycin) in 10 cm dishes and culture overnight at 37 &deg;C in a 5% CO2 incubator. Replace the medium with 5 ml of pre-warmed Opti-MEM I Reduced Serum Media (Invitrogen).</li>
	<li>Prepare Solution A: 10 &mu;g of shRNA-containing lentiviral vector, 5 &mu;g each of the third generation lentivirus packaging plasmids (prepared with high purity; the three packaging plasmids include VSV-G: for an envelope protein, pRSV-Rev: for rev, and pMDLg/pRRE: for gag/pol, which are essential for lentivirus packaging), 36 &mu;l of 2M CaCl2, and 300 &mu;l of sterile water.</li>
	<li>Prepare Solution B: 300 &mu;l of 2&times; Hepes Buffered Saline (281 mM NaCl, 100 mM HEPES, 1.5 mM Na2HPO4, pH adjusted to 7.12 by 0.5 N NaOH and sterilized by 0.22 &mu;m filter).</li>
	<li>Slowly drop Solution A into Solution B while bubbling air through Solution B with a pipette. Leave the tube at ambient temperature for 30 min.</li>
	<li>Slowly drop the mixed solutions A/B into the HEK 293T cell culture dish prepared in step 2.1 and incubate at 37 &deg;C in a 5% CO2 incubator for 4-6 h.</li>
	<li>Replace the medium with 7 ml of complete DMEM medium. After 24 h, add an additional 5 ml of the complete DMEM medium. Harvest the medium containing the lentivirus after another 24 h of culture.</li>
	<li>Spin the medium at 1,500 rpm, ambient temperature for 10 min. Filter the supernatant using a 0.45 &mu;m filter and spin the flow-through medium containing the lentivirus by ultracentrifugation at 25,000 rpm (equal to 125,000 &times;g with a SW28 swinging bucket rotor, Beckman Ultracentrifuge XL-90), 4 &deg;C for 90 min. Decant the supernatant to a container with 5% bleach (final concentration).</li>
	<li>Resuspend the lentivirus pellet in 0.5-1.0 ml of cold PBS and make lentivirus aliquots into 50-100 &mu;l per tube. Store them at -80 &deg;C.</li>
	<li>The titer of the lentivirus can be determined by commercial kits (such as QuickTiter Lentivirus Quantitation Kit from Cell Biolabs, Inc.), by a real-time PCR protocol8, or by determining the co-expressed fluorescent marker using fluorescence microscopy or flow cytometry. Typically, one 10-cm culture dish can produce about 0.5-1.0&times;108 infectious units (IU).</li>
</ol>
3. Infection and Characterization of Infected Cells
<ol>
	<li>Seed the cells to be infected in 2 ml of complete medium in a 6-well plate with 30-40% confluency (about 5-8 &times; 105 cells, depending on cell sizes). Culture the cells overnight at 37 &deg;C in a 5% CO2 incubator.</li>
	<li>Replace the medium with 1 ml of fresh medium containing 8 &mu;g/ml of polybrene (Sigma, Cat# H9268) or 5 &mu;g/ml of protamine (Sigma, Cat# P4020).</li>
	<li>Determine a suitable multiplicity of infection (MOI) range9 for a particular cell line using lentivirus with fluorescent marker. Calculate or empirically determine the amount of lentivirus to be used to infect the cell line. Slowly drop the lentivirus into the plate and gently shake it for 10 sec.</li>
	<li>Incubate the plate at 37 &deg;C in a 5% CO2 incubator for 6 h and then replace the medium with normal complete medium.</li>
	<li>At least two days after infection, check the cells using fluorescence microscopy for lentivirus expressing fluorescent markers and/or add the corresponding antibiotic to the medium for lentiviral vectors expressing antibiotic resistance genes. For an inducible vector, such as a Tet-On system (Figure 2), culture the cells in medium prepared with tetracycline-free FBS. Split the cells 2-3 days post infection. Take a portion of the cells to test inducible gene knockdown by culturing them in medium supplied with and without 1.5 &mu;g/ml doxycycline (Dox) for &ge;2 days.</li>
	<li>Check the gene knockdown by Western blot, Real-Time PCR or immunostaining &ge;2 days after infection, 2 days after antibiotic selection, or (for a Tet-On inducible system) &ge; 2 days after Dox addition.</li>
	<li>Utilize different approaches (such as cell proliferation assays, soft agar culture studies, migration assays, invasion assays and tumor formation studies) to determine the effects of the target gene knockdown on the malignancy of the cells.</li>
</ol>
4. Xenograft Mouse Model Study
<ol>
	<li>Clean all surfaces for mouse anesthetization and injection by 70% ethanol to prevent infection of mice. Anesthetize athymic nude mice (NCI-Frederick) using 2% isoflurane mixed with oxygen in an anesthesia induction chamber 10 min prior to cell inoculation. Maintain the anesthesia using 1% isoflurane mixed with oxygen through a nose cone. Carry out this step in a room with excellent ventilation and attach isoflurane scavenger filters to the induction chamber.</li>
	<li>Propagate the cells carrying shRNAs. Use tetracycline-free medium for Tet-On inducible vectors. Trypsinize the cells and resuspend them in complete medium containing 50% Matrigel. Position the nude mice on a heating pad (30 &deg;C) and inject 200 &mu;l of the cells (1-10 &times; 106, depending on the malignancy of the cells) into the mice with 1-2 injection sites per mouse. Use syringes with 25.5-gauge needles for subcutaneous injections, and 28-30 gauge needles for orthotopic injections.</li>
	<li>For constitutive shRNA vectors, utilize 2 groups of mice injected with the cells expressing the target gene shRNA and a scrambled shRNA. For Tet-On inducible shRNA vectors, utilize 4 groups of mice injected with cells carrying the target gene shRNA and a scrambled shRNA, supplied with normal water and Dox (1.5 mg/ml) containing water (2 shRNAs &times; 2 treatments = 4 groups). Replace the Dox-containing water twice a week.</li>
	<li>Monitor the length and width of the tumors weekly or biweekly by a digital vernier caliper and calculate tumor volumes (V) by the formula V = 1/2(length &times; width2)10. Ensure that tumor volume does not exceed the guidelines of the institutional ACUC (such as, 10% of the body weight). Euthanize any mouse using a protocol approved by the institutional ACUC if it shows any sign of extensive ulceration or necrosis, obvious infection, uncontrolled bleeding or end-stage illness.</li>
	<li>Tumor growth and metastasis can be monitored for the inoculated tumor cells expressing a bioluminescent marker11, such as Firefly luciferase. Clean all surfaces for mouse anesthetization and imaging by 70% ethanol to prevent infection of mice. Anesthetize the mice as described in step 4.1. Inject luciferin (150 mg/kg) intraperitoneally. Place the mice into a disinfected imaging chamber of a commercial imaging system, such as an IVIS Imaging System (Caliper Life Sciences, Inc.). Turn on the anesthesia lever to maintain anesthesia by 1% isoflurane mixed with oxygen. Conduct this step in a room with excellent ventilation.</li>
	<li>Take the first image by exposing 5 min and check its signal intensity. Adjust the exposure time to obtain images with optimal signal versus background. Determine the imaging time empirically, which is generally 1-5 min for subcutaneous tumors.</li>
	<li>Euthanize the mice by a protocol approved by the institutional ACUC when tumor sizes reach about 1,000 mm3, or as specified by the ACUC guidelines. Excise the xenograft tumors, weigh them and take pictures. Process the tumor samples for different studies, such as pathological analyses to determine tumor cell morphology, and Western blot and Real-Time PCR studies to test gene expression.</li>
</ol>
5. Representative Results
This protocol was used to study the effects of YY1 knockdown on xenograft tumor formation of Firefly luciferase- expressing MDA-MB-231 cells (human breast adenocarcinoma cells; Caliper Life Sciences) in athymic nude mice. The shRNA target sequence of human YY1 is "GGG AGC AGA AGC AGG TGC AGA T". A scrambled sequence "GGG ACT ACT CTA TTA CGT CAT T" was also created for a control (cont) shRNA, which did not have significant similarity to any known transcript. The oligonucleotides used to make Tet-On inducible shRNA constructs, with YY1 as an example, are shown in Table 1. As a result, two lentiviral vectors, pLu-Puro-Indu-YY1 shRNA and pLu-Puro-Indu-cont shRNA, were constructed and they were used to produce lentiviruses. We used these lentiviruses to individually infect two MDA-MB-231 cell clones (clones 1 and 3) expressing both tetracycline regulator (tetR) and Firefly luciferase. Polyclonal cell populations were obtained after puromycin selection. Figure 3A shows Dox-induced YY1 knockdown in these MDA-MB-231 cells infected by pLu-Puro-Indu-YY1 shRNA lentivirus. We then used the polyclonal cells of clone 3 infected individually by these inducible YY1 shRNA and cont shRNA lentiviruses and observed that YY1 depletion reduced invasiveness of MDA-MB-231 cells (Figure 3B). Western blot analyses confirmed Dox-induced YY1 silencing in MDA-MB-231 cells with the indu-YY1 shRNA, while the cells containing indu-cont shRNA did not show this effect (Figure 3C). We then used these cells for the xenograft mouse model study. Compared to the control groups, the mice implanted by the MDA-MB-231 cells with indu-YY1 shRNA and supplied with Dox-containing water showed reduced tumor formation, when visualized by bioluminescence (Figure 4A) and determined by tumor weights (Figure 4B). YY1 silencing in these xenograft tumors was confirmed by Western blot studies shown in Figure 4C.
<img alt="Figure 1" src="/files/ftp_upload/4129/4129fig1.jpg" /> 
Figure 1. Schematic diagram for the generation of a shRNA construct and shRNA transcription. The primers P1 to P6 are shown (see Table 1 for example sequences). Pol III: RNA polymerase III. (d): digested end. Drawing is not to scale. <a href="https://www.jove.com/files/ftp_upload/4129/4129fig1large.jpg" target="_blank"> Click here to view larger image</a>.
<img alt="Figure 2" src="/files/ftp_upload/4129/4129fig2.jpg" /> 
Figure 2. Schematic diagrams of (A) a lentiviral vector and (B) the mechanism of the Tet-On inducible H1 promoter used for gene silencing. The lentiviral vector was reconstructed based on pLL3.714 . H1-Pr: H1 promoter; Ubc-Pr: Human ubiquitin C promoter; Puro: puromycin; TRE: Tetracycline responsive element; Dox: doxycycline. TetR: tetracycline regulator. 5'LTR, 3'SIN-LTR and WRE are essential components of a lentiviral vector14 .
<img alt="Figure 3" src="/files/ftp_upload/4129/4129fig3.jpg" /> 
Figure 3. Dox-induced YY1 silencing and its effect on invasiveness of MDA-MB-231 cells. A. YY1 levels in two polyclonal cell populations derived from TetR-expressing clones 1 and 3 infected by pLu-Puro-Indu-YY1 shRNA lentivirus and cultured in the absence and presence of Dox. B. Boyden chamber assay of MDA-MB-231 cells with inducible shRNAs. (* P &lt; 0.05 versus other three groups). C. Representative Western blots of YY1 expression in these four cell populations.
 
<img alt="Figure 4" src="/files/ftp_upload/4129/4129fig4.jpg" /> 
Figure 4. Effects of induced YY1 silencing on xenograft tumor formation by MDA-MB-231 cells. A. Schematic diagram of cell implantation (left) and representative bioluminescent images captured by the IVIS Imaging System at 4 weeks (right) post cell inoculation. B. Xenograft tumor weights at 4 weeks. * P &le; 0.05. C. Western blots of YY1 and &beta;-actin expression in xenografts of MDA-MB-231 cells with indu-YY1 shRNA in the absence and presence of Dox. Labels on the top are the names of individual mice.
<table border="1">
	<tbody>
		<tr>
			<td>Oligonucleotide</td>
			<td>Sequence (5' - 3')</td>
			<td>Usage and location</td>
		</tr>
		<tr>
			<td>P1 (Tet-On H1)</td>
			<td>cagt GGATCC CGA ACG CTG ACG TCA TCA ACC C</td>
			<td>PCR; at 5'-end of the H1 promoter</td>
		</tr>
		<tr>
			<td>P1 (U6)</td>
			<td>cagt GGATCC GAC GCC GCC ATC TCT AGG</td>
			<td>PCR; at 5'-end of the U6 promoter</td>
		</tr>
		<tr>
			<td>P2 (for YY1 with Tet-On H1)</td>
			<td>cagc AAGCTT GAA atc tgc acc tgc ttc tgc tcc c GGG ATC TCT ATC ACT GAT AGG GAA C</td>
			<td>PCR; at 3'-end of the Tet-On inducible H1 promoter</td>
		</tr>
		<tr>
			<td>P2 (for YY1 with U6)</td>
			<td>cagc AAGCTT GAA atc tgc acc tgc ttc tgc tcc c aaa caa ggc ttt tct cca agg gat a</td>
			<td>PCR; at 3'-end of the U6 promoter</td>
		</tr>
		<tr>
			<td>P3 (for YY1)</td>
			<td>agc tt atc tgc acc tgc ttc tgc tcc c ttttt g</td>
			<td>To be annealed with P4</td>
		</tr>
		<tr>
			<td>P4 (for YY1)</td>
			<td>aattc aaaaa ggg agc aga agc agg tgc aga t a</td>
			<td>To be annealed with P3</td>
		</tr>
		<tr>
			<td>P5 (for pLL3.7)</td>
			<td>ggg tac agt gca ggg gaa aga ata g</td>
			<td>For PCR screening, used with P6</td>
		</tr>
		<tr>
			<td>P6 (for Tet-On H1)</td>
			<td>GAT TTC CCA GAA CAC ATA GCG AC</td>
			<td>For PCR screening, used with P5</td>
		</tr>
		<tr>
			<td>P6 (for U6)</td>
			<td>AGG GTG AGT TTC CTT TTG TGC TG</td>
			<td>For PCR screening, used with P5</td>
		</tr>
	</tbody>
</table>
Table 1. Synthesized oligonucleotides used in generating shRNA constructs. P1 and P6 sequences for the mouse U6 and Tet-On inducible human H1 promoters are provided. Human YY1 is used as an example with a shRNA target sequence of GGG AGC AGA AGC AGG TGC AGA T. The sequences specific to YY1 are highlighted. Two P2 sequences of YY1 are designed for the two promoters, respectively. The constitutive U6/YY1 shRNA was described previously15, while the Tet-On H1/YY1 was used in this protocol. P5 is vector-specific and the sequence for pLL3.714 is shown. The sequences of restriction enzymes are underlined, while the sequences to anneal to the promoters during PCR amplification are italicized.

<ol>
	<li>Elbashir, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplexes+of+21-nucleotide+RNAs+mediate+RNA+interference+in+cultured+mammalian+cells.">Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.</a> Nature. 411, 494-498 (2001).</li><li>Sui, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+vector-based+RNAi+technology+to+suppress+gene+expression+in+mammalian+cells.">A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5515-5520 (2002).</li><li>Brummelkamp, T. R., Bernards, R., Agami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+stable+expression+of+short+interfering+RNAs+in+mammalian+cells.">A system for stable expression of short interfering RNAs in mammalian cells.</a> Science. 296, 550-553 (2002).</li><li>Zhang, Q., Stovall, D. B., Inoue, K., Sui, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oncogenic+role+of+Yin+Yang+1.">The oncogenic role of Yin Yang 1.</a> Crit. Rev. Oncog. 16, 163-197 (2011).</li><li>Wan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yin+Yang+1+plays+an+essential+role+in+breast+cancer+and+negatively+regulates+p27.">Yin Yang 1 plays an essential role in breast cancer and negatively regulates p27.</a> Am. J. Pathol. , (2012).</li><li>Sui, G., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+by+a+DNA+vector-based+RNAi+technology.">Gene silencing by a DNA vector-based RNAi technology.</a> Methods in Molecular Biology. 309, 205-218 (2005).</li><li>Trower, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+PCR-based+colony+screening+protocol+for+cloned+inserts.">A rapid PCR-based colony screening protocol for cloned inserts.</a> Methods Mol. Biol. 58, 329-333 (1996).</li><li>Lizee, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+reverse+transcriptase-polymerase+chain+reaction+as+a+method+for+determining+lentiviral+vector+titers+and+measuring+transgene+expression.">Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.</a> Hum. Gene Ther. 14, 497-507 (2003).</li><li>Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+controlled+conditions+in+lentiviral+vector+titration+and+in+the+use+of+multiplicity+of+infection+(MOI)+for+predicting+gene+transfer+events.">The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events.</a> Genet Vaccines Ther. 2, 6 (2004).</li><li>Geran, R. I., Greenberg, N. H., MacDonald, M. M., Schumacher, A. M., Abbott, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+screening+chemical+agents+and+natural+products+against+animal+tumors+and+other+biological+systems.">Protocols for screening chemical agents and natural products against animal tumors and other biological systems.</a> Cancer Chemother. Rep. 3, 1-103 (1972).</li><li>Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+in+living+subjects:+seeing+fundamental+biological+processes+in+a+new+light.">Molecular imaging in living subjects: seeing fundamental biological processes in a new light.</a> Genes Dev. 17, 545-580 (2003).</li><li>Inoue, H., Nojima, H., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+Escherichia+coli+with+plasmids.">High efficiency transformation of Escherichia coli with plasmids.</a> Gene. 96, 23-28 (1990).</li><li>Toledo, J. R., Prieto, Y., Oramas, N., Sanchez, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine-based+transfection+method+as+a+simple+and+effective+way+to+produce+recombinant+lentiviral+vectors.">Polyethylenimine-based transfection method as a simple and effective way to produce recombinant lentiviral vectors.</a> Appl. Biochem. Biotechnol. 157, 538-544 (2009).</li><li>Rubinson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lentivirus-based+system+to+functionally+silence+genes+in+primary+mammalian+cells,+stem+cells+and+transgenic+mice+by+RNA+interference.">A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.</a> Nat Genet. 33, 401-406 (2003).</li><li>Sui, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yin+Yang+1+is+a+negative+regulator+of+p53.">Yin Yang 1 is a negative regulator of p53.</a> Cell. 117, 859-872 (2004).</li></ol>

No conflicts of interest declared.

This protocol describes a method to knock down a gene using shRNA and visualize its biological effect using bioluminescent imaging of tumor cell growth in vivo. The target site of a shRNA should not contain any of the three restriction sites (BamHI, HindIII and EcoRI) used for subcloning. In a rare scenario when any of these sites is present, an additional restriction enzyme can be used to replace it in generating the construct.
Several key steps in this protocol will speed up ...

In addition to common laboratory equipment used for RNA and protein analyses and cell culture, the following materials and reagents are required for this protocol.
<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 </tr>
 <tr>
 <td>Biosafety Cabinet suitable for Biosafety Level 2 containment</td>
 </tr>
 <tr>
 <td>PCR thermocycler</td>
 </tr>
 <tr>
 <td>Ultracentrifuge</td>
 </tr>
 <tr>
 <td>Anesthesia-induction chamber with isoflurane scavengers</td>
 </tr>
 <tr>
 <td>Digital Vernier caliper</td>
 </tr>
 <tr>
 <td>IVIS imaging system</td>
 </tr>
 <tr>
 <td>Highly competent DH5a E. coli</td>
 </tr>
 <tr>
 <td>EcoRI, BamHI, & HindIII restriction enzymes</td>
 </tr>
 <tr>
 <td>T4 DNA Ligase</td>
 </tr>
 <tr>
 <td>Third generation lentivirus packaging plasmids VSV-G, pRSV-Rev and pMDLg/pRRE</td>
 </tr>
 </tbody>
</table>
Materials List

dna vector-based rna interference to study gene function in cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

Watch this Scientific Journal Video about DNA Vector-based RNA Interference to Study Gene Function in Cancer at JoVE.com

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

dna-vector-based-rna-interference-to-study-gene-function-in-cancer

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Medicine

20.3K Views.  Wake Forest University School of Medicine. RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

Video: DNA Vector-based RNA Interference to Study Gene Function in Cancer

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...