Name: engineering artificial factors to specifically manipulate alternative splicing in human cells
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Description: Watch this Scientific Journal Video about Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells at JoVE.com

This work was supported by NIH grant R01-CA158283 and NSFC grant 31400726 to Z.W. Y.W. is funded by the Young Thousand Talents Program and the National Natural Science Foundation of China (grants 31471235 and 81422038). X.Y. is funded by the postdoctoral science foundation of China (2015M571612).

1. Construction of a PUF Scaffold with Customized RNA-binding Specificity by Overlapping PCR
<ol>
	<li>Design a series of PCR primers containing the PUF sequences that specifically recognize different RNA nucleotides in each position12 (see Table 1 for the primer sequences and see Figure 1A for the RNA:PUF recognition code). For each PUF repeat, design four different primers to recognize a different base on each position. 
	NOT...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+regulation:+from+a+parts+list+of+regulatory+elements+to+an+integrated+splicing+code.">Splicing regulation: from a parts list of regulatory elements to an integrated splicing code.</a> Rna. 14 (5), 802-813 (2008).</li><li>Matera, A. G., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+day+in+the+life+of+the+spliceosome.">A day in the life of the spliceosome.</a> Nat Rev Mol Cell Biol. 15 (2), 108-121 (2014).</li><li>Singh, R. K., Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-mRNA+splicing+in+disease+and+therapeutics.">Pre-mRNA splicing in disease and therapeutics.</a> Trends Mol Med. 18 (8), 472-482 (2012).</li><li>Wang, G. -. S., Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+in+disease:+disruption+of+the+splicing+code+and+the+decoding+machinery.">Splicing in disease: disruption of the splicing code and the decoding machinery.</a> Nat Rev Genet. 8 (10), 749-761 (2007).</li><li>Li, Y. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+splicing+is+a+primary+link+between+genetic+variation+and+disease.">RNA splicing is a primary link between genetic variation and disease.</a> Science. 352 (6285), 600-604 (2016).</li><li>Scotti, M. M., Swanson, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+mis-splicing+in+disease.">RNA mis-splicing in disease.</a> Nat Rev Genet. 17 (1), 19-32 (2016).</li><li>Black, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+alternative+pre-messenger+RNA+splicing.">Mechanisms of alternative pre-messenger RNA splicing.</a> Annu Rev Biochem. 72 (1), 291-336 (2003).</li><li>Graveley, B. R., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine/serine-rich+domains+of+SR+proteins+can+function+as+activators+of+pre-mRNA+splicing.">Arginine/serine-rich domains of SR proteins can function as activators of pre-mRNA splicing.</a> Mol cell. 1 (5), 765-771 (1998).</li><li>Del Gatto-Konczak, F., Olive, M., Gesnel, M. -. C., Breathnach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hnRNP+A1+recruited+to+an+exon+in+vivo+can+function+as+an+exon+splicing+silencer.">hnRNP A1 recruited to an exon in vivo can function as an exon splicing silencer.</a> Mol Cell Biol. 19 (1), 251-260 (1999).</li><li>Auweter, S. D., Oberstrass, F. C., Allain, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-specific+binding+of+single-stranded+RNA:+is+there+a+code+for+recognition.">Sequence-specific binding of single-stranded RNA: is there a code for recognition.</a> Nucleic Acids Res. 34 (17), 4943-4959 (2006).</li><li>Dong, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+and+modular+binding+code+for+cytosine+recognition+in+Pumilio/FBF+(PUF)+RNA-binding+domains.">Specific and modular binding code for cytosine recognition in Pumilio/FBF (PUF) RNA-binding domains.</a> J Biol Chem. 286 (30), 26732-26742 (2011).</li><li>Filipovska, A., Razif, M. F., Nyg&#229;rd, K. K., Rackham, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+universal+code+for+RNA+recognition+by+PUF+proteins.">A universal code for RNA recognition by PUF proteins.</a> Nat Chem Biol. 7 (7), 425-427 (2011).</li><li>Wei, H., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+RNA-binding+proteins+with+diverse+activities.">Engineering RNA-binding proteins with diverse activities.</a> Wiley Interdiscip Rev RNA. 6 (6), 597-613 (2015).</li><li>Wang, Y., Cheong, C. -. G., Hall, T. M. T., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+splicing+factors+with+designed+specificities.">Engineering splicing factors with designed specificities.</a> Nat Methods. 6 (11), 825-830 (2009).</li><li>Choudhury, R., Tsai, Y. S., Dominguez, D., Wang, Y., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+RNA+endonucleases+with+customized+sequence+specificities.">Engineering RNA endonucleases with customized sequence specificities.</a> Nat Commun. 3, 1147 (2012).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complex+network+of+factors+with+overlapping+affinities+represses+splicing+through+intronic+elements.">A complex network of factors with overlapping affinities represses splicing through intronic elements.</a> Nat Struct Mol Biol. 20 (1), 36-45 (2013).</li><li>McCabe, B. C., Gollnick, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+levels+of+trp+RNA-binding+attenuation+protein+in+Bacillus+subtilis.">Cellular levels of trp RNA-binding attenuation protein in Bacillus subtilis.</a> J Bacteriol. 186 (15), 5157-5159 (2004).</li><li>Wang, Y., Ma, M., Xiao, X., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intronic+splicing+enhancers,+cognate+splicing+factors+and+context-dependent+regulation+rules.">Intronic splicing enhancers, cognate splicing factors and context-dependent regulation rules.</a> Nat Struct Mol Biol. 19 (10), 1044-1052 (2012).</li><li>Choudhury, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+splicing+activator+DAZAP1+integrates+splicing+control+into+MEK/Erk-regulated+cell+proliferation+and+migration.">The splicing activator DAZAP1 integrates splicing control into MEK/Erk-regulated cell proliferation and migration.</a> Nat Commun. 5, (2014).</li><li>Xiao, X., Wang, Z., Jang, M., Burge, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coevolutionary+networks+of+splicing+cis-regulatory+elements.">Coevolutionary networks of splicing cis-regulatory elements.</a> Proc Natl Acad Sci U S A. 104 (47), 18583-18588 (2007).</li><li>Wang, Z., Xiao, X., Van Nostrand, E., Burge, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+and+specific+functions+of+exonic+splicing+silencers+in+splicing+control.">General and specific functions of exonic splicing silencers in splicing control.</a> Mol Cell. 23 (1), 61-70 (2006).</li><li>Li, M., Husic, N., Lin, Y., Snider, 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=Production+of+lentiviral+vectors+for+transducing+cells+from+the+central+nervous+system.">Production of lentiviral vectors for transducing cells from the central nervous system.</a> J Vis Exp. (63), e4031 (2012).</li><li>Tiscornia, G., Singer, O., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+purification+of+lentiviral+vectors.">Production and purification of lentiviral vectors.</a> Nat Protoc. 1 (1), 241-245 (2006).</li><li>Venables, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+and+alternative+splicing+in+cancer.">Aberrant and alternative splicing in cancer.</a> Cancer Res. 64 (21), 7647-7654 (2004).</li><li>Cooke, A., Prigge, A., Opperman, L., Wickens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+translational+regulation+using+the+PUF+protein+family+scaffold.">Targeted translational regulation using the PUF protein family scaffold.</a> Proc Natl Acad Sci U S A. 108 (38), 15870-15875 (2011).</li><li>He, C., Zhou, F., Zuo, Z., Cheng, H., Zhou, 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+global+view+of+cancer-specific+transcript+variants+by+subtractive+transcriptome-wide+analysis.">A global view of cancer-specific transcript variants by subtractive transcriptome-wide analysis.</a> PloS one. 4 (3), 4732 (2009).</li><li>Venables, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+alternative+splicing+markers+for+breast+cancer.">Identification of alternative splicing markers for breast cancer.</a> Cancer Res. 68 (22), 9525-9531 (2008).</li><li>Shapiro, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+EMT-Driven+Alternative+Splicing+Program+Occurs+in+Human+Breast+Cancer.">An EMT-Driven Alternative Splicing Program Occurs in Human Breast Cancer.</a> PLoS Genet. 7 (8), 1002218 (2011).</li><li>David, C. J., Manley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+pre-mRNA+splicing+regulation+in+cancer:+pathways+and+programs+unhinged.">Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged.</a> Genes Dev. 24 (21), 2343-2364 (2010).</li><li>Ladomery, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+alternative+splicing+is+another+hallmark+of+cancer.">Aberrant alternative splicing is another hallmark of cancer.</a> Int J Cell biol. 2013, (2013).</li><li>Karni, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gene+encoding+the+splicing+factor+SF2/ASF+is+a+proto-oncogene.">The gene encoding the splicing factor SF2/ASF is a proto-oncogene.</a> Nat Struct Mol Biol. 14 (3), 185-193 (2007).</li><li>Anczuk&#242;w, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SRSF1-regulated+alternative+splicing+in+breast+cancer.">SRSF1-regulated alternative splicing in breast cancer.</a> Mol cell. 60 (1), 105-117 (2015).</li></ol>

This report provides a detailed description for the design and construction of artificial splicing factors that can specifically manipulate the alternative splicing of a target gene. This method takes advantage of the unique RNA binding mode of PUF repeats to produce an RNA-binding scaffold with customized specificity. It can be used to either activate or repress splicing.
The critical step in this protocol is the generation of the reprogramed PUF domain that defines the specificity of ESFs. A...

Most human genes undergo Alternative Splicing (AS) to produce multiple isoforms with distinct activities, which has greatly increased the coding complexity of the genome1,2. AS provides a major mechanism to regulate gene function, and it is tightly regulated through diverse pathways in different cellular and developmental stages3,4. Because splicing misregulation is a common cause of human disease5,6,7,8, targeting splicing regulation is becoming an attractive therapeutic route.
According to a simplified model of splicing regulation, AS is mainly controlled by Splicing Regulatory cis-Elements (SREs) in pre-mRNA that function as splicing enhancers or silencers of alternative exons. These SREs specifically recruit various trans-acting protein factors (i.e.&#160;splicing factors) that promote or suppress the splicing reaction3,9. Most trans-acting splicing factors have separate sequence-specific RNA binding domains to recognize their targets and effector domains to control splicing. The best-known examples are the members of the serine/arginine-rich (SR) protein family that contain N-terminal RNA Recognition Motifs (RRMs), which bind exonic splicing enhancers, and C-terminal RS domains, which promote exon inclusion10. Conversely, hnRNP A1 binds to exonic splicing silencers through the RRM domains and inhibits exon inclusion through a C-terminal glycine-rich domain11. Using such modular configurations, researchers should be able to engineer artificial splicing factors by combining a specific RNA-Binding Domain (RBD) with different effector domains that activate or inhibit splicing.
The key of such a design is to use an RBD that recognizes given targets with programmable RNA binding specificity, which is analogous to the DNA-binding mode of the TALE domain. However, most native splicing factors contain RRM or K Homology (KH) domains, which recognize short RNA elements with weak affinity and thus lack a predictive RNA-protein recognition &#34;code&#34;12. The RBD of PUF repeat proteins (i.e.&#160;the PUF domain) has a unique RNA recognition mode, allowing for the redesign of PUF domains to specifically recognize different RNA targets13,14. The canonical PUF domain contains eight repeats of three &#945;-helices, each recognizing a single base in an 8-nt RNA target. The side chains of amino acids at certain positions of the second &#945;-helix form specific hydrogen bonds with the Watson-Crick edge of the RNA base, which determines the RNA binding specificity of each repeat (Figure 1A). The code for RNA base recognition of the PUF repeat is surprisingly simple (Figure 1A), allowing for the generation of PUF domains that recognize any possible 8-base combination (reviewed by Wei and Wang15).
This modular design principle allows for the generation of an Engineered Splicing Factor (ESF) that consists of a customized PUF domain and a splicing modulation domain (i.e.&#160;an SR domain or a Gly-rich domain). These ESFs can function as either splicing activators or as inhibitors to control various types of splicing events, and they have proven useful as tools to manipulate the splicing of endogenous genes related to human disease16,17. As an example, we have constructed PUF-Gly-type ESFs to specifically alter the splicing of the Bcl-x gene, converting the anti-apoptotic long isoform (Bcl-xL) to the pro-apoptotic short isoform (Bcl-xS). Shifting the ratio of the Bcl-x isoform was sufficient to sensitize several cancer cells to multiple anti-cancer chemotherapy drugs16, suggesting that these artificial factors may be useful as potential therapeutic reagents.
In addition to controlling splicing with known splicing effector domains (e.g., an RS or Gly-rich domain), the engineered PUF factors can also be used to examine the activities of new splicing factors. For example, using this approach, we have demonstrated that the C-terminal domain of several SR proteins can activate or inhibit splicing when binding to different pre-mRNA regions18, that the alanine-rich motif of RBM4 can inhibit splicing19, and that the proline-rich motif of DAZAP1 can enhance splicing20,21. These new functional domains can be used to construct additional types of artificial factors to fine-tune splicing.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">High-fidelity DNA polymerase (Phusion High-Fidelity) with PCR buffer</td>
			<td style="width:155px;">New England Biolabs</td>
			<td style="width:103px;">M0530L</td>
			<td style="width:491px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA ligase (T4 DNA ligase)</td>
			<td>New England Biolabs</td>
			<td>M0202L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liposomal transfection reagent (Lipofectamine 2000)</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reduced serum medium (Opti-MEM)</td>
			<td>Gibco</td>
			<td>31985-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNA extraction buffer (TRIzol Reagent)</td>
			<td>ambion</td>
			<td>15596018</td>
			<td>TRIzol reagent includes phenol, which can cause burns. Wear gloves when handling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA (Bovine Serum Albumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A7638-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS (1X)</td>
			<td>Life Technologies</td>
			<td>10010-031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperScript III reverse transcriptase</td>
			<td>Invitrogen</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Caspase-3 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9668</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PARP&#160;antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bcl-x antibody</td>
			<td>BD Bioscience</td>
			<td>610211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">beta-actin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>A5441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">alpha-tubulin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>T5168</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FLAG antibody</td>
			<td>Sigma-Aldrich</td>
			<td>F4042</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose membrane</td>
			<td>Amersham-Pharmacia</td>
			<td>RPN203D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECL Western Blotting detection reagents</td>
			<td>Invitrogen</td>
			<td>WP20005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy5-dCTP</td>
			<td>GE Healthcare</td>
			<td>PA55021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence-activated cell sorter</td>
			<td>BD Bioscience</td>
			<td>FACSCalibur&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>GE Healthcare</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propidium iodide (PI)</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7638-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton-X100</td>
			<td>Promega</td>
			<td>H5142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-lysine&#160;</td>
			<td>Sigma</td>
			<td>P-4832</td>
			<td>Filter sterilize and store at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vector&#160; pWPXLd</td>
			<td>Addgene</td>
			<td>12258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vector pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vector psPAX2</td>
			<td>Addgene</td>
			<td>12260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I (RNase-free)</td>
			<td>New England Biolabs</td>
			<td>M0303S</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Oligo(dT)18 Primer</td>
			<td>Thermo Scientific</td>
			<td>SO131&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse secondary antibody (Anti-mouse IgG, HRP-linked Antibody)</td>
			<td>Cell Signaling Technology</td>
			<td>7076S</td>
			<td></td>
		</tr>
	</tbody>
</table>

engineering artificial factors to specifically manipulate alternative splicing in human cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes the complete protocol for the design and construction of ESFs and splicing reporters. It also outlines the further application of ESFs in manipulating the AS of endogenous genes16. To illustrate typical results of ESF-mediated splicing changes, we use the data from our previous work as an example. The ESFs with different functional domains can be used to promote or inhibit the inclusion of the target cassette exon (Figure ...

Watch this Scientific Journal Video about Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells at JoVE.com

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

The overall goal of this protocol is to engineer artificial splicing factors that can specifically manipulate alternative splicing of a given target. This method can help to answer key questions in the field of RNA processing, such as the regulation and the misregulation of alternate splicing in cancer cells. The main advantage of this technique is its flexibility.We can design artificial factors that either promote or inhibit different type of alternate splicing in any given genes. Demonstrating this procedure will be Huan-Huan, a research associate of my laboratory, and Qianyun, a postdoc in my laboratory. Wild-type PUF DNA is used as a template for generating the customized PUF domain scaffold.Use PCR primers containing PUF sequences that specifically recognize different RNA nucleotides in each position. For each PUF repeat, use four different primers to recognize a different base on each position. In round one PCR, use the primer pairs shown to generate coding sequences for four PUF repeats, universal bridge fragments, and cap fragments.In round two, use the PCR products generated in the last step as templates with the primer combinations shown to generate coding sequences for RNA recognition codes R-one to R-four, and R-five to R-eight. In round three, use the products from round two and bridge four to five as templates with the R-one forward primer and R-eight reverse primer to generate coding sequences for R-one to R-eight without caps. In round four PCR, use R-one to R-eight, five-prime end cap and three-prime end cap as templates, to generate coding sequences for complete PUF domains.To begin transfection of HEK-293T cells, mix the liposomal transfection reagent by gently inverting the bottles. Then dilute two microliters of liposomal transfection reagent in 50 microliters of reduced serum medium per transfection. Mix gently, and then incubate for five minutes at room temperature.Next, dilute 04 micrograms of glycine-rich domain expression vector, and 0.2 micrograms of the modular splicing reporter plasmid in 50 microliters of reduced serum medium in a sterile tube. Then dilute 0.4 micrograms of RS-effector domain expression vector, and 0.2 micrograms of PGZ-three reporter plasmid in 50 microliters of reduced serum medium. Lastly, dilute 0.4 micrograms of PGL-RS-PUF expression vector and 0.2 micrograms of PEZ-1B or PEZ-2F reporter plasmids in 50 microliters of reduced serum medium in a sterile tube.After five minutes of incubation at room temperature, gently mix the diluted liposomal transfection reagent with the diluted plasmid mixtures. Incubate the final mixtures for 20 minutes at room temperature. Then add the transfection mixtures containing the expression vectors to each well of a 24-well plate seeded with HEK-293 cells, and incubate for at least 12 hours in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide.After 12 hours of incubation, harvest the transfected cells by trypsinization and centrifugation. Following centrifugation, discard the medium and add 0.5 milliliters of RNA extraction buffer per tube. Add 0.1 milliliters of chloroform per 0.5 milliliters of RNA extraction buffer to each sample.Following the incubation, centrifuge the tubes for 15 minutes at 12, 000 times G and four degrees Celsius. Then transfer the aqueous base to a fresh tube. Add 0.25 milliliters of isopropanol per 0.5 milliliters of RNA extraction buffer used in the initial homogenization.Mix by vortexing, and incubate at room temperature for 10 minutes. After a centrifugation as before, the RNA precipitate is usually visible on the bottom of the tube. Discard the supernatant, and wash the RNA pellet with 0.5 milliliters of 75%ethanol per 0.5 milliliters of RNA extraction buffer.Vortex vigorously, and then centrifuge at 7, 500 times G for five minutes at four degrees Celsius. After the spin, remove the supernatant, and air-dry the RNA pellets. Dissolve the RNAs in 50 microliters of RNase-free water.Next, add two microliters of five units per microliter DNase-one, seven microliters of 10X buffer, and 11 microliters of water to each 50-microliter RNA solution. After the incubation, heat the solutions to 70 degrees Celsius for 15 minutes to inactivate the DNase. After performing a reverse transcriptase reaction to synthesize cDNA from each sample, add the components of the body labeled PCR reaction in the order shown on screen to PCR tubes.Prepare one reaction per sample. Run the RT-PCR program on the thermal cycler. Finally, resolve the PCR products by electrophoresis through a 10%polyacrylamide gel with TBE buffer.Use a fluorescence scanner to visualize the products, and measure the amount of each spliced isoform using densitometry software. For the immunofluorescence assay to measure apoptosis, prepare a transfection mix containing glycine-rich domain expression plasmids as previously shown, and transfect HeLa cells grown on polylysine-coated glass cover slips in a six-well plate. 24 hours after transfection, fix the cells on the cover slips with one milliliter of 4%paraformaldehyde in 1x PBS for 20 minutes at room temperature.Then gently wash the cells on the cover slips with two milliliters of 1x PBS for five minutes. Next, permeabilize the cells with 0.2%Triton X-100 in 1x PBS for 10 minutes. After washing three times with 1x PBS as before, block non-specific binding with 3%bovine serum albumin in 1x PBS for 10 minutes.Dilute the flag antibody one to 1, 000 in 3%BSA in PBS, and pipette 30 microliters of the diluted flag antibody onto parafilm sheet. Use forceps to remove a cover slip. Carefully dry off excess buffer with lab wipes, and then place the cover slip cell-side down on the anti-flag solution.Incubate for one hour at room temperature. Following the incubation, add approximately 500 microliters of 1x PBS to the cell-side of the cover slip until it floats on top of the solution. Return it to the six-well plate with 1x PBS in the wells.Incubate with secondary antibody on parafilm as before for 15 minutes. Wash as before, and then mount the cover slips on microscope slides using mounting medium containing DAPI. Visualize the cells using a fluorescence microscope, and photograph them using a digital camera.Using immunofluorescence microscopy, many cells expressing Gly-PUF-531 had fragmented nuclear DNA, indicating that they were undergoing apoptosis. As a control, cells expressing Gly-PUF wild-type had less fragmented nuclear DNA when compared to Gly-PUF-531 transfected cells. Here, immunofluorescence microscopy using the anti-flag antibody merged with DAPI-stained cells shows that the Gly-PUF-531 localized predominantly in the nuclei of transfected cells.Gly-PUF wild-type also localized in the nuclei of transfected cells, suggesting that these ESFs dissociate from their targets when the fully-spliced mRNAs are transported into the cytoplasm. The percentage of apoptotic cells with fragmented nuclear DNA was measures from randomly chosen fields of fluorescence microscopy images. Once mastered, the construction of PUF scaffolds with RNA binding specificity can be done in two days if it is performed properly.After watching this video, you should have a good understanding of how to specifically manipulate alternative splicing in human cells by engineering artificial factors.

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Research

JoVE Journal

Genetics

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3&#697; end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5&#697; untranslated region (5&#697; UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein up-regulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.

SINEUPs are synthetic antisense non-coding RNAs, which contain a binding domain (BD) and an effector domain (ED) and up-regulate translation of target mRNA. Here, we describe detection methods for SINEUPs in cultured cell lines, analysis of their translation-promoting activity by Western-blot and a semi-automated high throughput imaging system.

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, the data obtained from rodent islets are often not fully reproduced in or applicable to human islets due to well-known differences in islet structure and function between the species. Currently, techniques that are available to manipulate gene expression of human islets are very limited. Introduction of transgene into intact islets by adenovirus, plasmid, and oligonucleotides often suffers from low efficiency and high toxicity. Low efficiency is especially problematic in gene downregulation studies in intact islets, which require high efficiency. It has been known that enzymatically-dispersed islet cells reaggregate in culture forming spheroids termed pseudoislets. Size-controlled reaggregation of human islet cells creates pseudoislets that maintain dynamic first phase insulin secretion after prolonged culture and provide a window to efficiently introduce lentiviral short hairpin RNA (shRNA) with low toxicity. Here, a detailed protocol for the creation of human pseudoislets after lentiviral transduction using two commercially available multiwell plates is described. The protocol can be easily performed and allows for efficient downregulation of genes and assessment of dynamism of insulin secretion using human islet cells. Thus, human pseudoislets with lentiviral mediated gene modulation provide a powerful and versatile model to assess gene function within human islet cells.

A protocol to create gene modified human pseudoislets from dispersed human islet cells that are transduced by lentivirus carrying short hairpin RNA (shRNA) is presented. This protocol utilizes readily available enzyme and culture vessels, can be performed easily, and produces genetically modified human pseudoislets suitable for functional and morphological studies.

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...