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Endogenous Reporter Cells: Gold Standard
  • Xactagen has developed technology that makes reporter cells a more powerful tool for understanding gene expression in biological research and drug discovery. Sensitivity gains provide indexing of up to 50 percent more genes for in vitro and in vivo applications while production advances provide greater access to whole-genome, gene-specific, pathway-specific, or drug-inducible reporter cells.

  • Endogenous reporter cells are the gold standard for reporter cells. By introducing a reporter into an endogenous gene, all promoters, enhancers and silencers for the endogenous gene are utilized, including those residing tens of thousands of base pairs from the transcription start site. Local chromosomal influences are also captured, such as DNA methylation. Plus, with Xactagen’s endogenous reporter cells, the mRNA transcripts retain exon sequences including the 3’ untranslated region (3’ UTR) which is often the target of micro-RNAs.

  • In a direct comparison of exogenous reporter cells versus endogenous reporter cells, Yan et. al. (2009) demonstrated that gene-targeted (endogenous) reporter cells consistently provided predictive indexing of gene expression while exogenous expression was more variable, and in some cases unable to index regulation of gene expression. Yan, Z., Lei-Butters, D., Engelhardt, J.F., and Leno, G.H. Indexing TNF-alpha gene expression using a gene-targeted reporter cell line. BMC Biol. 2009 Feb 16; 7:8.


Endogenous Reporter Cells: Whole-Genome Libraries
    Production

  • To produce whole-genome libraries of endogenous reporter cells, a proprietary gene trap vector is transfected into cells where it then inserts into an endogenous gene (Figure 1). Expression of the reporter gene is then under regulation of regulatory elements for that gene. The integrated vector provides for transcription of a fusion mRNA comprised of 5’ gene sequences, vector sequences, and 3’ gene sequences. An internal ribosome entry site is used to translate the fusion mRNA into luciferase (reporter) and neo (selectable marker). Elements of the vector have been optimized for reporter expression and vector retention over multiple cell generations. Tens of thousands of reporter cells can be produced. As part of production, Xactagen uses either inverse PCR or plasmid rescue technologies combined with DNA sequencing to determine vector insertion sites and to associate each cell in the library with a specific gene or transcription unit.


Figure 1: Gene trap production of endogenous reporter cells.


    Gene Representation

  • Gene representation in reporter cell libraries made with Xactagen’s gene trapping technology is very large. It is estimated that perhaps 50% of human genes (~12,000 out of 20,000 to 25,000 genes in the human genome) are expressed in any given cell. As indicated in Table 1, a representative gene trap library prepared in HCT116 human colon cancer cells is comprised of ~13,000 known or predicted genes, suggesting that most if not all expressed genes are represented in the library. Genes within diverse gene families are represented as are genes within most chromosomal loci (see Table 2). Surprisingly, about 48% of transcription units (~12,000 transcription units) in Xactagen’s reporter cell libraries represent unknown or unpredicted DNA transcripts. At least some of the unknown/unpredicted transcription units have structures indicative of micro-RNAs. Still others appear within genes, but utilizing the non-coding DNA strand. The functions of many of these unknown/unpredicted transcription units await further research.




Endogenous Reporter Cells: Induction Cloned Libraries
    Production

  • One of the most efficient means for reporter cell production offered by Xactagen is mediated by induction cloning. Induction cloning allows researchers to acquire sets of genes that are induced or suppressed by cellular ligands, hormones, cytokines, peptides, siRNAs, miRNAs, antibodies, chemical agents, or dug products/candidates directed at specific targets or signal transduction pathways.

  • For production, Xactagen begins by randomly introducing Xactagen’s gene trap vectors into the genomes of recipient cells to produce tens of thousands of reporter cells. Rather than directly determining the insertion sites for all members of this library (as is done for whole-genome production: see above), clones are first subjected to stimulation (ligand, hormones, cytokine, peptide, siRNA, miRNA, antibody, compound, drug) and a reporter assay performed. Clones whose reporter activity is increased or decreased are then expanded and insertion sites determined by inverse PCR or plasmid rescue combined with sequencing to associate specific genes or other transcription units with each cell clone.


Endogenous Reporter Cells: Gene Targeting
    Production

  • Xactagen has been highly successful at directing reporter vector insertion into specific genes by gene targeting. Gene targeting vectors are produced by flanking Xactagen’s gene trap vectors with 1 to 2 kb of DNA sequences homologous to desired insertion sites in genes. The recombination vectors are then introduced into recipient cells where the flanking sequences help to direct integration to the desired site. Vector design greatly assists with selection of cells containing gene targeted insertions as opposed to cells that have integrated vector randomly into the genome. Gene targeting frequency as high as 50% of antibiotic resistant cells has been attained.


Endogenous Reporter Cells: Reporter Cell Sensitivity
    Distribution of Reporter Activities in Cloned Cells

  • As demonstrated above, Xactagen’s gene trap vectors capture the majority of genes expressed in a particular cell type. To determine the level of reporter expression in these clones relative to background (e.g. signal to noise), an Xactagen gene trap reporter vector using membrane-anchored Gaussia luciferase was introduced into HCT116 colon cancer cells and a panel of 96, randomly selected G418 resistant cells was expanded for analysis. Gaussia luciferase flash activity was measured for each clone (figure 2). Data is represented relative to luciferase activity in parental HCT116 cells (lacking Gaussia luciferase). Data was sorted from lowest expressing clones to highest expressing clones.


Figure 2: Reporter activities in HCT116 human colon cancer cells

  • The results indicate that eighty eight out of the ninety-six clones had expression levels greater than 5-fold background. Median activity was 30-fold background. The highest level observed for these particular 96 clones was 1,263-fold background. In other experiments with other clones, expression levels up to ~208,000-fold background have been attained (see Properties: Performance Data, Figure 4).

  • We conclude that not only are Xactagen vectors capable of trapping most if not all genes expressed in any particular cell, but expression levels for the majority of these exceed 5-fold noise. Given that Xactagen reporter vectors and cells are 50 to 1,000-fold more sensitive than other reporter systems (see Properties: Data Performance), and the median activity with Xactagen reporter vectors was ~ 30 fold background, we further conclude that up to 50% of genes expressed in any particular cell may be below detection level (signal to noise) of other reporter vectors/systems.


  • Gene Trap Efficiency of Xactagen Vectors

  • Gene trap vectors rely upon endogenous promoters to drive expression of the reporter gene as well as neo, for selection of stable transfectants on G418. Accordingly, we predicted that only Xactagen gene trap vectors would provide sufficient expression to drive neo expression if introduced into weakly expressed transcription units/genes.

To test this prediction, we compared gene trapping efficiencies between two early versions of Xactagen gene trap vectors (XGEN1, XGEN2), Xactagen’s current gene trap vector design (XGEN3), and a conventional vector not utilizing Xactagen design features (Conventional). Transfections were performed by electroporation followed by selection of stable transfectants with G418. Colonies per transfection were recorded (Figure 3: done in qualdruplicate).

The results demonstrate that Xactagen vectors produce from 2-fold to up to 4-fold more G418 resistant colonies than vectors with conventional components. Because all vectors were approximately the same size and were prepared using identical reagents and procedures, it is unlikely that differences in transfection efficiency account for differences in colony production. Rather, the data are consistent with our hypothesis: more colonies are attained due to the ability of Xactagen vectors, but not conventional vectors, to trap and express sufficient neo when integrated into weakly expressed transcription units/genes.


Figure 3: Gene trap efficiencies of Xactagen vectors versus conventional vectors.



Standard "exogenous" Reporter Cells
    Integration of Xactagen Technology into Standard "Exogenous" Vectors

  • Standard “exogenous” reporter vectors are ideal for transient transfections and are well suited for studies with stable transfections under certain conditions. Synthesized or PCR-generated promoters, enhancers and silencers are typically introduced into promoterless vector backgrounds (Figure 4). The resulting vectors are then introduced into cells where assays are performed shortly after transfection (transient transfections) or after selection for stable integration into the host genome (stable transfections). Xactagen vectors are especially well-suited, due to engineered reporter genes, vector design, optimized assay reagents and ease of use (see Properties for details). Like endogenous reporter cells, these unique elements combine to enable analysis of not only strong promoters, but also weak promoters typically not measurable with competitive products.

  • Although applicable for many purposes, Standard “exogenous” Reporter Vectors have limits for indexing gene expression. First, they do not retain ALL gene elements (enhancers, silencers) that combine to regulate normal gene expression. Enhancers that reside tens of thousands of base-pairs from the transcription start site will not typically be retained. Second, local chromosomal influences such as DNA methylation will not be retained. And third, for stable transfectants, regulatory elements in the recipient cell’s genomic DNA that reside close to the insertion site of the vector can influence gene expression. It is therefore desirable to characterize exogenous reporter cells prior to use to confirm they are functioning properly.


  • Vectors for Transient or Stable Transfections

  • Xactagen offers two kinds of reporter vectors: bi-cistronic vectors and mono-cistronic vectors (Figure 4).


Figure 4: Bi-cistronic and Mono-cistronic Vectors.

  • Bi-cistronic Vectors: For bi-cistronic vectors, introduced promoters/enhancers/silencers drive expression of a bi-cistronic mRNA comprised of a reporter gene and neo (e.g. two translational units on one mRNA). Translation of the reporter gene uses the 5’CAP of the mRNA to assemble ribosomes. Translation of neo uses an internal ribosome entry site (IRES) for assembly of ribosomes. Because there are no other promoter/enhancers introduced into the vector, bi-cistronic vectors minimize the potential for promoter/enhancer cross talk and they are preferred (default) vectors for transient transfections. In many cases, bicistronic vectors may also be used to derive stable transfectants: due to vector designs that optimize expression, even weak promoters can provide sufficient expression of neo for selection on G418.

  • Mono-cistronic Vectors: For mono-cistronic vectors, introduced promoters/enhancers/silencers drive expression of the reporter gene while an SV40 promoter/enhancer drives expression of neo for selection on G418 (e.g. each gene has its own promoter). They are preferred vectors for stable transfections when bi-cistronic vectors can not be used.


  • Xactagen Gaussia Luciferase Assay

    Higher Sensitivity and Ease of Use
    Xactagen has engineered Gaussia luciferase for sensitivity and ease of use for bench top applications and high throughput screening (HTS). In vitro, Xactagen’s assay reagents provide for higher signal, less noise and more consistent and accurate indexing of gene expression. For more details, see Properties. In vivo, Gaussia luciferase reporters have been shown to be more active than firefly or Renilla luciferases: the increase in reporter activity of Gaussia luciferase more than compensates for less efficient transmittance due to blue-green luminescence versus red luminescence charateristic of firefly luciferase.


    Figure 5: Schematic of assay steps for analysis of Gaussia luciferase activity.