We thank all members of our laboratory for helpful discussions. We thank Joanna Lisztwan and Christina Gewinner for critical reading of the manuscript. This work was supported by the BBSRC grant BB/I021248/1 and the Wellcome Trust grant 090090/Z/09/Z.A.H. is a Wellcome Trust Research Career Development fellow at the UCL Cancer Institute.

1. Cloning of pcDNA6_TetR_IRES_blast
<ol>
	<li>As illustrated in Figure 3, perform a partial digest of the pcDNA6/TR plasmid (V1025-20, Invitrogen) with the restriction enzymes XbaI and NcoI to remove the TetR gene and the promoter of the blasticidin resistance (BlastR) gene. Isolate the 4.6 kb vector fragment.</li>
	<li>To remove the polyadenylation sequence from the TetR gene, introduce by PCR an XhoI site immediately after the Stop codon of the TetR cDNA while conserving the XbaI site at the 5'end of the cDNA. Subclone the resulting fragment into pMigR117 using restriction enzymes XbaI and XhoI to generate the pMigR1_TetR plasmid.</li>
	<li>Digest the pMigR1_TetR vector with the restriction enzymes XbaI and NcoI to release the TetR_IRES fragment. Isolate the 1.25 kb insert fragment.</li>
	<li>Ligate the 4.6 kb pcDNA6/TR and the 1.25 kb TetR_IRES fragments. Transform the ligation product into competent E.coli and identify the desired pcDNA6_TetR_IRES_blast construct using standard molecular biology techniques.</li>
</ol>
2. Generation of Cell Lines Stably Expressing TetR
<ol>
	<li>On day 1, Seed 1x106 cells per 10-cm tissue culture plate using your standard growth medium (e.g. DMEM supplemented with 10% FCS). Seed two plates, one for transfection with pcDNA6_TetR_IRES_blast, and the other as a negative control for blasticidin selection. Ensure to use the lowest passage possible and the recommended growth medium.</li>
	<li>On day 2, transfect one plate with 2 &mu;g of pcDNA6_TetR_IRES_blast using Fugene 6 (E2691, Promega) following the manufacturer's instructions. Do not transfect the second plate.</li>
	<li>On day 3, add standard growth medium containing 5 &mu;g/ml blasticidin to both plates. Please note that the optimal selection concentration may vary for a given cell line and might need to be determined beforehand.</li>
	<li>On day 4, split the cells at 1/5, 1/25, 1/50 and 1/100 using 10-cm plates and standard growth medium containing 5 mg/ml blasticidin. Make sure to properly label plates containing untransfected or transfected cells. Prepare two plates per dilution step.</li>
	<li>Check cells daily, ensuring that all cells on the untransfected plates have died within the first week. Change selection media every 2-3 days.</li>
	<li>After 1-2 weeks, blasticidin-resistant colonies should begin to appear. Select plates containing 5 to 50 colonies to ensure that single colonies can be picked.</li>
	<li>When colonies have reached a diameter of approximately 5 mm (or bigger), isolate at least 12 independent clones using cloning cylinders to pick single colonies. Transfer each clone into a separate well of a 24-well tissue culture plate. When confluent, split the cells from each well into one single well of a 6-well tissue culture plate.</li>
	<li>Continue to culture clones in selection media. When confluent, split cells from each well into two single wells of a 6-well tissue culture plate.</li>
	<li>For each clone to be tested, freeze one well of confluent cells and harvest cells in the other well for subsequent testing by immunoblotting.</li>
	<li>Detect TetR by Western blotting using the mouse monoclonal anti-TET02 antibody (MoBiTec GmbH). Remember to include a lysate of the parental cell line as a negative control.</li>
	<li>Select 3 clones with the highest TetR expression and expand each clonal culture. Freeze stocks of each promising clone as soon as possible. Finally, examine the molecular and cell biological parameters of interest by comparing the parental cell lines with the TetR-expressing clones. Select the 'best' clone with the highest TetR expression that does not display any alterations in the molecular and cell biological parameters of interest.</li>
</ol>
3. Pilot Testing of pTER and pT-Rex DEST30 Vectors Expressing shRNA or cDNAs, Respectively
<ol>
	<li>Generate pTER plasmid with shRNA inserts as described12. Construct pT-Rex DEST30 vector (12301-016, Invitrogen) containing the cDNA of interest using Gateway technology (Invitrogen). In case of cDNA expression, addition of a tag to the cDNA will later facilitate detection of exogenously expressed protein.</li>
	<li>Transiently transfect the parental target cell line with pTER or pT-Rex DEST30 plasmids (expressing either shRNAs or cDNAs of interest) using the transfection reagent of choice. For testing of shRNA expression plasmids, ensure high transfection efficiencies can be achieved.</li>
	<li>Harvest transfected cells and process for immunoblotting using the appropriate antibodies, expecting to detect a depletion or overexpression of your gene of interest.</li>
</ol>
4. Generation of Stable Cell Lines with Tetracycline-inducible (Tet-on) Expression of shRNAs or cDNAs Using pTER or pT-Rex DEST30 Vectors
<ol>
	<li>On day 1, seed 1x106 cells of the 'best' TetR-expressing clone per 10-cm plate using your standard growth medium supplemented with certified Tetracycline-free serum (for example FBS from Invitrogen; 16000-014). Seed one plate for transfection with pTER (or pT-Rex DEST30), and one plate as negative control for double selection. Make sure to use the lowest passage possible.</li>
	<li>On day 2, transfect one plate with 2 &mu;g of pTER (or pT-Rex DEST30) using Fugene 6. Do not transfect the second plate.</li>
	<li>On day 3, add standard growth medium containing 5 &mu;g/ml blasticidin and 500 &mu;g/ml zeocin (or 1 mg/ml G418).</li>
	<li>On day 4, split the cells at 1/5, 1/25, 1/50 and 1/100 using 10-cm plates and standard growth medium containing 5 &mu;g/ml blasticidin and 500 &mu;g/ml zeocin (or 1 mg/ml G418). Prepare two plates per dilution step.</li>
	<li>Check the cells daily, making sure that within the first week all cells on the untransfected plates have died. Change the standard growth medium containing blasticidin and zeocin (or G418) every 3-4 days.</li>
	<li>After 2-3 weeks, blasticidin/zeocin (or blasticidin/G418) double-resistant colonies should begin to appear. Select plates containing 5 to 50 colonies to pick single colonies.</li>
	<li>When colonies have reached a diameter of approximately 5 mm (or bigger), use cloning cylinders to pick single colonies. Isolate at least 24 individual clones. Transfer each clone into a separate well of a 24-well tissue culture plate.</li>
	<li>Culture clones in medium containing maintenance concentrations of blasticidin (5 &mu;g/ml) and zeocin (250 &mu;g/ml) [or G418 (0.5 mg/ml)]. When confluent, split the cells from each well into one single well of a 6-well tissue culture plate.</li>
	<li>For each clone to be tested, split one confluent well of a 6-well plate into three single wells of a 6-well plate. When confluent, freeze cells from one well. Use the remaining two wells for testing the tetracycline-inducible expression. Add tetracycline at a concentration of 1 &mu;g/ml to one well, while leaving the second well tetracycline free.</li>
	<li>After 24 to 96 hr of incubation with Tetracycline, harvest cells and process for immunoblotting using appropriate antibodies. Shorter tetracycline incubation times are recommended when screening for the induction of cDNA expression, while longer tetracycline treatments will be necessary to determine shRNA-mediated depletion of endogenous proteins.</li>
	<li>Select at least two clones with the desired depletion/overexpression and expand each culture. Freeze stocks of each promising clone as soon as possible. When growing Tet-on clones for experiments always make sure to use media supplemented with Tetracycline-free serum and the respective selection antibiotics at maintenance concentrations.</li>
</ol>

<ol>
	<li>Postle, K., Nguyen, T. T., Bertrand, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence+of+the+repressor+gene+of+the+TN10+tetracycline+resistance+determinant.">Nucleotide sequence of the repressor gene of the TN10 tetracycline resistance determinant.</a> Nucleic Acids Res. 12, 4849-4863 (1984).</li><li>Gossen, M., Bujard, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+control+of+gene+expression+in+mammalian+cells+by+tetracycline-responsive+promoters.">Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.</a> Proc. Natl. Acad. Sci. U.S.A. 89, 5547-5551 (1992).</li><li>Gossen, M., 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=Transcriptional+activation+by+tetracyclines+in+mammalian+cells.">Transcriptional activation by tetracyclines in mammalian cells.</a> Science. 268, 1766-1769 (1995).</li><li>Elbashir, S. M., 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=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>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>McManus, M. T., Petersen, C. P., Haines, B. B., Chen, J., Sharp, P. A. <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+using+micro-RNA+designed+hairpins.">Gene silencing using micro-RNA designed hairpins.</a> RNA. 8, 842-850 (2002).</li><li>Miyagishi, M., Taira, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U6+promoter-driven+siRNAs+with+four+uridine+3'+overhangs+efficiently+suppress+targeted+gene+expression+in+mammalian+cells.">U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells.</a> Nat. Biotechnol. 20, 497-500 (2002).</li><li>Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., Conklin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNAs+(shRNAs)+induce+sequence-specific+silencing+in+mammalian+cells.">Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells.</a> Genes Dev. 16, 948-958 (2002).</li><li>Paul, C. P., Good, P. D., Winer, I., Engelke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+expression+of+small+interfering+RNA+in+human+cells.">Effective expression of small interfering RNA in human cells.</a> Nat. Biotechnol. 20, 505-508 (2002).</li><li>Sui, G., 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+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>Yu, J. Y., DeRuiter, S. L., Turner, 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=RNA+interference+by+expression+of+short-interfering+RNAs+and+hairpin+RNAs+in+mammalian+cells.">RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 6047-6052 (2002).</li><li>van de Wetering, M., 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+inhibition+of+gene+expression+using+a+stably+integrated,+inducible+small-interfering-RNA+vector.">Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector.</a> EMBO Rep. 4, 609-615 (2003).</li><li>Hergovich, A., 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+MST1+and+hMOB1+tumor+suppressors+control+human+centrosome+duplication+by+regulating+NDR+kinase+phosphorylation.">The MST1 and hMOB1 tumor suppressors control human centrosome duplication by regulating NDR kinase phosphorylation.</a> Curr. Biol. 19, 1692-1702 (2009).</li><li>Hergovich, A., Lamla, S., Nigg, E. A., Hemmings, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome-associated+NDR+kinase+regulates+centrosome+duplication.">Centrosome-associated NDR kinase regulates centrosome duplication.</a> Mol. Cell. 25, 625-634 (2007).</li><li>Kohler, R. S., Schmitz, D., Cornils, H., Hemmings, B. A., Hergovich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+NDR/LATS+interactions+with+the+human+MOB+family+reveal+a+negative+role+for+human+MOB2+in+the+regulation+of+human+NDR+kinases.">Differential NDR/LATS interactions with the human MOB family reveal a negative role for human MOB2 in the regulation of human NDR kinases.</a> Mol. Cell Biol. 30, 4507-4520 (2010).</li><li>Vichalkovski, A., 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=NDR+kinase+is+activated+by+RASSF1A/MST1+in+response+to+Fas+receptor+stimulation+and+promotes+apoptosis.">NDR kinase is activated by RASSF1A/MST1 in response to Fas receptor stimulation and promotes apoptosis.</a> Curr. Biol. 18, 1889-1895 (2008).</li><li>Pear, W. 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=Efficient+and+rapid+induction+of+a+chronic+myelogenous+leukemia-like+myeloproliferative+disease+in+mice+receiving+P210+bcr/abl-transduced+bone+marrow.">Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow.</a> Blood. 92, 3780-3792 (1998).</li></ol>

No conflicts of interest declared.

We believe that Tet-on systems greatly facilitate the analysis of gene function, particularly in cell systems that are difficult to manipulate and/or when the manipulated gene is essential for cell survival. Furthermore, the ability to control gene expression by highly specific Tet-on systems offers the opportunity to study gene functions at different stages (for example during cell cycle progression or well-defined differentiation processes). The method presented here will enable researchers to generate the desir...

<table><tbody><tr><td>Fetal Bovine Serum(FBS)</td><td>Invitrogen</td><td>16000-044</td><td>Tested Tet-free</td></tr><tr><td>Blasticidin</td><td>Invivogen</td><td>ant-bl-1</td><td> </td></tr><tr><td>Zeocin</td><td>Invivogen</td><td>ant-zn-5</td><td> </td></tr><tr><td>G418</td><td>PAA laboratories</td><td>P31-011</td><td>100 mg/ml in media</td></tr><tr><td>anti-TET02</td><td>MoBiTec GmbH</td><td>TET02</td><td>Use at 1/1000 to 1/2000 for WB</td></tr><tr><td>pcDNA6/TR</td><td>Invitrogen</td><td>V1025-20</td><td> </td></tr><tr><td>pT-Rex DEST30</td><td>Invitrogen</td><td>12301-016</td><td> </td></tr><tr><td>Tetracycline</td><td>Sigma</td><td>87128</td><td>2 mg/ml in ethanol</td></tr><tr><td>Doxycycline</td><td>Sigma</td><td>D9891</td><td>2 mg/ml in water</td></tr><tr><td>Cloning cylinders</td><td>Bellco Glass Inc.</td><td>2090-00808</td><td>re-useable</td></tr></tbody></table>

generation of stable human cell lines with tetracycline-inducible (tet-on) shrna or cdna expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

An example for the initial characterization of RPE-1 cell lines stably expressing TetR is shown in Figure 4. Note that all RPE-1 clones express varying levels of TetR (compare lanes 2 and 5), while the parental cell line (which serves as negative control) does not express the exogenous TetR protein (Figure 4, lane 1). This variation in TetR expression among RPE-1 Tet-on cell clones is expected, since the expression of the TetR expressing plasmids is highly dependent on the plasmid...

Watch this Scientific Journal Video about Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression at JoVE.com

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

generation-stable-human-cell-lines-with-tetracycline-inducible-tet-on

Research

JoVE Journal

Biology

35.2K Views.  UCL Cancer Institute. Een snelle en eenvoudige manier om humane cellijnen met induceerbare en omkeerbare cDNA overexpressie of shRNA-gemedieerde knock-down van het gen van belang genereren. Deze methode kunnen onderzoekers betrouwbaar en zeer reproduceerbaar manipuleren cellijnen die moeilijk te veranderen door transiënte transfectie methoden of conventionele knockdown / knockout strategieën.

Video: Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Genetics

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Cancer Research

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs&#160;biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

Immunology and Infection

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in C2C12 myoblasts can be applied to study the role of these proteins in skeletal muscle development. Here, we describe a protocol to deplete the ECM protein ADAMTSL2 as an example, using small-hairpin (sh) RNA in C2C12 cells. Following transfection of shRNA plasmids, stable cells were batch-selected using puromycin. We further describe the maintenance of these cell lines and the phenotypic analysis via mRNA expression, protein expression, and C2C12 differentiation. The advantages of the method are the relatively fast generation of stable C2C12 knockdown cells and the reliable differentiation of C2C12 cells into multinucleated myotubes upon depletion of serum in the cell culture medium. Differentiation of C2C12 cells can be monitored by bright field microscopy and by measuring the expression levels of canonical marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC) indicating the progression of C2C12 myoblast differentiation into myotubes. In contrast to the transient knockdown of genes with small-interfering (si) RNA, genes that are expressed later during C2C12 differentiation or during myotube maturation can be targeted more efficiently by generating C2C12 cells that stably express shRNA. Limitations of the method are a variability in the knockdown efficiencies, depending on the specific shRNA that may be overcome by using gene knockout strategies based on CRISPR/Cas9, as well as potential off-target effects of the shRNA that should be considered.

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin shRNA

We provide a protocol to stably knock down genes encoding extracellular matrix (ECM) proteins in C2C12 myoblasts using small-hairpin (sh) RNA. Targeting ADAMTSL2 as an example, we describe the methods for the validation of the knockdown efficiency on the mRNA, protein, and cellular level during C2C12 myoblast to myotube differentiation.

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Short Hairpin RNA-Mediated Gene Knockdown in iHSPCs In Vitro: A Lentivirus-Based shRNA Expression System Delivery into iHSPCs for Knockdown of Specific Gene Expression

Source: Hsiao, Y. L., et al. <a href="https://www.jove.com/v/62730">Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro.</a> J. Vis. Exp. (2022).
In this video, we demonstrate a method to perform transduction of shRNA lentiviral vectors to obtain stable knockdown cell lines in immortalized hematopoietic stem and progenitor cells (iHSPCs).

Short Hairpin RNA Mediated Gene Knockdown in iHSPCs In Vitro: A Lentivirus-Based ShRNA Expression System Delivery into iHSPCs for Knockdown of Specific Gene Expression and Generation of Stable Knockdo

Source: Hsiao, Y. L., et al.  Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

Samenvatting

Meer video's verkennen