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<section class="row help-vector-system-detail" ng-controller="VectorSystemCtrl">
<article class="col-xs-4 content-left">
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<header class="menu-heading">
<span class="header-node">Mammalian Gene Expression Vectors</span>
<span><i
class="fa fa-chevron-up pull-right"></i></span>
<span><i
class="fa fa-chevron-down pull-right hide"></i></span>
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<h4>All-Purpose Gene Expression</h4>
<div>
<i class="fs20">• </i><a
class="active"
href="/learning-center/vector-system/pRP_Ex_plus.html">
Regular plasmid
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class=""
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Lentivirus
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class=""
href="/learning-center/vector-system/pMXs_Ex.html">
MMLV retrovirus
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<i class="fs20">• </i><a
class=""
href="/learning-center/vector-system/pMSCV_Exp.html">
MSCV retrovirus
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<i class="fs20">• </i><a
class=""
href="/learning-center/vector-system/pAV_Ex_plus.html">
Adenovirus
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<i class="fs20">• </i><a
class=""
href="/learning-center/vector-system/pAAV_Ex.html">
AAV
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<i class="fs20">• </i><a
class=""
href="/learning-center/vector-system/pPB_Ex.html">
PiggyBac
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<i class="fs20">• </i><a
class=""
href="/learning-center/vector-system/pTol2_Ex.html">
Tol2
</a>
</div>
</li>
<li>
<h4>Inducible Gene Expression (Tet Based)</h4>
<div class="third-nav"><i class="fs20">• </i>Regular plasmid (Tet-On)</div>
<div class="pl17">
<i class="fs20">• </i><a
class=""
href="/learning-center/vector-system/pRP_Tet_On.html">
Regular tet-on vector</a>
</div>
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<li>
<h4>Conditional Gene Expression (Cre-Lox Based)</h4>
<div>
<i class="fs20">• </i><a
class=""
href="/learning-center/vector-system/pRP_Ex_LoxP_stop_LoxP.html">
Regular plasmid (LoxP-Stop-LoxP)
</a>
</div>
<div>
<i class="fs20">• </i><a
class=""
href="/learning-center/vector-system/pPB_LoxP_Stop_LoxP.html">
PiggyBac (LoxP-Stop-LoxP)
</a>
</div>
<div class="third-nav"><i class="fs20">• </i>Regular plasmid (FLEX)</div>
<div class="pl17">
<i class="fs20">• </i><a
class=""
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Cre-on gene expression vector</a>
</div>
<div class="pl17">
<i class="fs20">• </i><a
class=""
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Cre-off gene expression vector</a>
</div>
<div class="pl17">
<i class="fs20">• </i><a
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Cre-switch gene expression vector</a>
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Lentivirus
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Adenovirus
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Single-stranded AAV (ssAAV)</a>
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PiggyBac
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Lentivirus
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Single gRNA vector</a>
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<h4>gRNA Sensor Vector (for Testing gRNA Specificity)</h4>
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Regular plasmid
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Lentiviral msgRNA (for dCas9-SAM system)
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Regular plasmid (for enhancer testing)
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PiggyBac (for enhancer testing)
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Tol2
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<div class="third-nav"><i class="fs20">• </i>P-element transposon vector pUAST</div>
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<i class="fs20">• </i><a
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UAS-Hsp70 promoter</a>
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<div class="third-nav"><i class="fs20">• </i>att-site integration vector pUASTattB</div>
<div class="pl17">
<i class="fs20">• </i><a
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UAS-Hsp70 promoter</a>
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<div class="third-nav"><i class="fs20">• </i>Dual-integration vector pUASTB</div>
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UAS-Hsp70 promoter</a>
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<h4></h4>
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<i class="fs20">• </i><a
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T-DNA binary vector (for plant transformation)
</a>
</div>
<div>
<i class="fs20">• </i><a
class=""
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Regular plasmid (for electroporation of plant protoplasts)
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<h4>Bacteria</h4>
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pET
</a>
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pBAD
</a>
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Cold-shock induced
</a>
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<li>
<h4>Yeast</h4>
<div>
<i class="fs20">• </i><a
class=""
href="/learning-center/vector-system/pPp_Exp.html">
Pichia pastoris
</a>
</div>
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<i class="fs20">• </i><a
class=""
href="/learning-center/vector-system/pSC_Ex.html">
Saccharomyces cerevisiae
</a>
</div>
</li>
<li>
<h4>Insect</h4>
<div>
<i class="fs20">• </i><a
class=""
href="/learning-center/vector-system/pBV.html">
Baculovirus transfer vector (single promoter)
</a>
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<span class="header-node">In Vitro Transcription Vectors</span>
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In vitro transcription vector (for mRNA)
</a>
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<i class="fs20">• </i><a
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In vitro transcription vector (for in situ hybridization)
</a>
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<i class="fs20">• </i><a
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href="/learning-center/vector-system/pPp_T7_sRNA.html">
In vitro transcription vector (for small RNA)
</a>
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</article>
<article class="col-xs-8 content-body">
<div>
<h1 class="fs26 pink">
<p>Regular Plasmid Gene Expression Vector
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</h1>
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<h2 class="fs16"><strong>Overview</strong>
</h2>
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<p>Delivering plasmid vectors into mammalian cells by conventional transfection is one of the most widely used procedures in biomedical research. While a number of more sophisticated gene delivery vector systems have been developed over the years such as lentiviral vectors, adenovirus vectors, AAV vectors and piggyBac, conventional plasmid transfection remains the workhorse of gene delivery in many labs. This is largely due to its technical simplicity as well as good efficiency in a wide range of cell types. A key feature of transfection with regular plasmid vectors is that it is transient, with only a very low fraction of cells stably integrating the plasmid in the genome (typically less than 1%).</p>
<p>For further information about this vector system, please refer to the papers below.</p>
<table class="table table-bordered table-condensed table-hover table-striped">
<tbody>
<tr>
<th style="text-align: center;">References</th>
<th style="text-align: center;">Topic</th>
</tr>
<tr>
<td><a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=Mol+Biotechnol.+16%3A151+%282000%29" target="_blank" rel="nofollow noopener">Mol Biotechnol. 16:151 (2000)</a></td>
<td>Overview of vector design for mammalian gene expression</td>
</tr>
<tr>
<td><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC413492/" target="_blank" rel="nofollow noopener">EMBO J. 12:2539 (1993)</a></td>
<td>Transcription blocker prevent transcriptional interference</td>
</tr>
</tbody>
</table>
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<div class="content-box">
<h2 class="fs16"><strong><p><strong>Highlights</strong></p></strong></h2>
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Our vector is optimized for high copy number replication in E. coli and high-efficiency transfection. Cells transfected with the vector can be selected and/or visualized based on marker gene expression as chosen by the user.
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<h2 class="fs16"><strong><p><strong>Advantages</strong></p></strong></h2>
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<p><strong style="color:#000000">Technical simplicity:</strong> Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.</p> <p><strong style="color:#000000">Very large cargo space:</strong> Our vector can accommodate ~30 kb of total DNA. The plasmid backbone only occupies about 3 kb, leaving plenty of room to accommodate the user's sequence of interest.</p> <p><strong style="color:#000000">High-level expression:</strong> Conventional transfection of plasmids can often result in very high copy numbers in cells (up to several thousand copies per cell). This can lead to very high expression levels of the genes carried on the vector.</p>
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<div class="content-box">
<h2 class="fs16"><strong><p><strong>Disadvantages</strong></p></strong></h2>
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<p><strong style="color:#000000">Non-integration of vector DNA:</strong> Conventional transfection of plasmid vectors is also referred to as transient transfection because the vector stays mostly as episomal DNA in cells without integration. However, plasmid DNA can integrate permanently into the host genome at a very low frequency (one per 10<sup>2</sup> to 10<sup>6</sup> cells depending on cell type). If a drug resistance or fluorescence marker is incorporated into the plasmid, cells stably integrating the plasmid can be derived by drug selection or cell sorting after extended culture.</p><p><strong style="color:#000000">Limited cell type range:</strong> The efficiency of plasmid transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo.</p><p><strong style="color:#000000">Non-uniformity of gene delivery:</strong> Although a successful transfection can result in very high average copy number of the transfected plasmid vector per cell, this can be highly non-uniform. Some cells can carry many copies while others carry very few or none. This is unlike transduction by virus-based vectors which tends to result in relatively uniform gene delivery into cells.</p>
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<div class="content-box">
<h2 class="fs16"><strong>Key components</strong></h2>
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<div>
<p style="text-align: center;"><app-guide-vector symbol="pRP_Ex_plus"></app-guide-vector></p>
<p><strong style="color: #000000;">Promoter:</strong> The promoter that drives your gene of interest is placed here.</p>
<p><strong style="color: #000000;">Kozak:</strong> Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.</p>
<p><strong style="color: #000000;">ORF:</strong> The open reading frame of your gene of interest is placed here.</p>
<p><strong style="color: #000000;">SV40 late pA:</strong> Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.</p>
<p><strong style="color: #000000;">CMV promoter:</strong> Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of the downstream marker gene.</p>
<p><strong style="color: #000000;">Marker:</strong> A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.</p>
<p><strong style="color: #000000;">BGH pA:</strong> Bovine growth hormone polyadenylation. It facilitates transcriptional termination of the upstream ORF.</p>
<p><strong style="color: #000000;">pUC ori:</strong> pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.</p>
<p><strong style="color: #000000;">Ampicillin:</strong> Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.</p>
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