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Stable and strong promoter?

Stable and strong promoter?


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I need a mammalian promoter that will maintain stable expression through differentiation.

I was originally planning to employ UbC for this specific project, however new information from a different experiment has come to light and now I need a promoter that has both extreme stability and high strength (as you know, UbC is renowned for it's very low expression rates).

Do any of you have suggestions? Perhaps I should look into a hybrid promoter of sorts…

Thank you,

CDB


There seems to be quite an amount of resources to help OP.

Addgene has a page on topic with list and description of common promoters usable in variety of organisms: Plasmids 101: The Promoter Region

Experimental biophysics textbook mentions some useful promoters as well, or this review.

Surprisingly, there has been some quantitative analysis of promoter strength: Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter, as well as effort toward stronger synthetic (version of CMV) Synthetic design of strong promoters

My conclusion is that either viral, or CAG (based on chicken actin) promoters might be useful candidates. But one cannot expect ubiquitous and even expression in various tissues/cells types without fine-tuning expression cassette.


Stable and strong promoter? - Biology

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Graphical abstract

The fall armyworm, Spodoptera frugiperda, native to the Americas, is becoming a worldwide pest inflicting damage to multiple crops (Jing et al., 2019). S. frugiperda cell line, Sf9 was developed from the ovaries and the Sf21 selected from Sf9 have been widely used for production of recombinant proteins (Davis et al., 1993 McCall et al., 2005). Transient transfection of plasmid constructs into these cells (Chang et al., 2018 Shen et al., 2014), selection of stable cells from transfected cells (Fernandes et al., 2012 Kempf et al., 2002), and baculovirus expression vector system (BEVS) (Smith et al., 1983) are commonly used to produce recombinant proteins in these cells. Early viral gene promoters, OpIE1 (Theilmann and Stewart, 1991), OpIE2 (Theilmann and Stewart, 1992), and hr5/ie1 (Jarvis et al., 1996) are the most commonly used promoters in plasmid-based expression in S. frugiperda cells. However, lower expression levels of foreign proteins is a limitation in using these viral promoters (Chang et al., 2018 Zhao and Eggleston, 1999). Endogenous promoters were shown to support high levels of protein production in Drosophila melanogaster S2 cells (Angelichio et al., 1991). Therefore, highly active endogenous promoters from S. frugiperda could increase the expression of recombinant proteins in S. frugiperda cell lines. The highly active late viral gene promoters, p10 and polyhedrin support high levels of protein expression through the use of baculoviruses in S. frugiperda cells therefore, this system is widely used for expression of recombinant proteins (Hill-Perkins and Possee, 1990). However, expression of recombinant proteins occurs at the late stage of baculovirus infection when the protein synthesis machinery in the host cells has already been impaired (Schultz and Friesen, 2009), and this results in inefficient processing and post-translational modification of recombinant proteins (Jarvis et al., 1993 Jarvis and Summers, 1989 Schultz and Friesen, 2009). The highly active endogenous promoters may facilitate the expression of recombinant proteins during early stages of baculovirus infection resulting in improvements to processing and post-translational modification of recombinant proteins.

Promoters derived from host insects have been successfully used in driving transgene expression in model insects, including Tribolium castaneum (Eckermann et al., 2018 Lorenzen et al., 2002 Rylee et al., 2018), Aedes aegypti (Anderson et al., 2010 De Valdez et al., 2011 Li et al., 2017), and Bombyx mori (Sakai et al., 2016 Tamura et al., 2000 Xu et al., 2019), which helped with both basic and applied research in these insects. Several S. frugiperda promoters have been identified by transcriptome and genome analysis of Sf21 cells however, none of them outperformed the early viral promoter OpIE2 in Sf21 cells (Bleckmann et al., 2015). We previously identified several heat-inducible promoters active in Sf9 cells, and two of them were more robust than OpIE2 promoter but less active than hr5/ie1 promoter (Chen et al., 2020). To identify S. frugiperda promoters that are more active than commercially used viral promoters, we analyzed a set of highly expressed genes from the transcriptomes of two S. frugiperda cell lines and three tissues. The performance of potential promoters of highly expressed genes was evaluated in Sf9 and Sf17 cells. The promoters that showed higher activity than the commercially used promoter (hr5/ie1) were further evaluated by transiently and stably expressing transgenes in Sf9 cells and FAW using plasmid-based and baculovirus expression systems, respectively. These studies identified two highly active S. frugiperda promoters that would be useful for protein expression, genome editing and production of transgenic insects in FAW and other lepidopteran insects.


Results and Discussion

Screening of endogenous strong promoters from P. mendocina NK-01 via RNA-seq analysis and promoter prediction

For RNA-seq analysis, transcriptional level of a gene is positively correlated with RPKM value 26 . Through RNA-seq analysis of P. mendocina NK-01, transcriptional levels of all genes were ranked from high to low based on their RPKM values. The first 30 genes ranked by RPKM values were assumed to be highly active at the transcriptional level (Table S1). Thus, the upstream regions of the 30 genes with high RPKM values were selected as the detection targets for promoter prediction. Through further screening using an online promoter prediction software, 10 out of 30 candidate sequences were identified as the putative promoter sequences (Fig. S1) and selected for subsequent cloning and characterization (Table 1).

Cloning of strong promoters from P. mendocina NK-01

The promoter regions of the 10 highly expressed genes were PCR-amplified from the genomic DNA of P. mendocina NK-01. To obtain the intact promoter sequence of each of the 10 highly expressed genes, in this work, the entire intergenic region between the highly expressed gene and its upstream gene was selected as the target region to be cloned by PCR, except for the native ribosomal binding site (RBS). The results from DNA sequencing showed that the cloned DNA fragments coincided with the selected intergenic regions at the nucleotide level (data not shown).

Characterization of the cloned promoters via qPCR

To assess the strengths of the cloned promoters, the promoters sequences were fused to the 5′-end of the amplified gfp gene and then inserted into a broad-host-range cloning vector pBBR1MCS-2 able to replicate in various gram-negative bacteria 27 using homologous recombination (Fig. 1). qPCR was employed for the analysis of transcriptional levels of gfp under different promoters at different growth phases, i.e., early log-phase (6 h), post log-phase (12 h) and stationary phase (15 h) (Fig. S2). Among the 10 tested promoters, the transcriptional levels of the five promoters P4, P6, P9, P16 and P25 were much higher than that of lac promoter at different growth phases. Compared with lac promoter, the strongest promoter P4 showed a 36-fold increase in the transcriptional activity at the stationary phase (Fig. 2). When detecting with most of the cloned promoters, the transcriptional levels of reporter gene gfp varied significantly at different growth phases. The five strong promoters P4, P6, P9, P16 and P25 had higher transcriptional levels in post log-phase than stationary phase or early log-phase (Fig. S3). In contrast, relatively minor differences in transcriptional levels were detected with the five strong promoters between stationary phase and early log-phase (Fig. S3). The promoter P16 had a relatively stable transcriptional activity throughout the growth period (Fig. S3). In previous studies, different types of promoters including strong promoters, growth phase-dependent promoters and constitutive promoters have been well characterized 10,13 . Because of good system compatibility with the host cell, the selection of endogenous promoters may be more practical and purposeful for their applications in synthetic biology and metabolic engineering of the host itself.

Recombinant plasmids for promoter characterization with gfp as a reporter gene. (a) Recombinant plasmid with a lac promoter as a control. (b) Recombinant plasmids for characterizing the strengths of 10 selected endogenous promoters.

Characterization of the chosen promoters and lac promoter via qPCR analysis. Transcription of gfp gene under different promoters in P. mendocina NKU was quantified at different growth phases. 16S rDNA gene was used as internal reference. The relative transcription value of gfp gene under lac promoter was set as 1. Data represent the mean values ± standard deviations of triplicate measurements from three independent experiments. A Student’s t-test was performed between lac promoter and chosen promoters. * and ** indicate P < 0.05 and P < 0.01, respectively.

The promoters P17, P18, and P29 had high RPKM values in the RNA-seq analysis, but the promoters placed on plasmid exhibited lower transcriptional levels than lac promoter at any growth phase (Fig. 2). The chosen endogenous promoters function well in the genome, which might be attributed to the assistance of the nearby regulatory sequences. Once the promoters were cloned separately, they did not work well.

Since the screened endogenous promoters were expected to be used for improving PHA production, the RNA-seq data were obtained with P. mendocina grown in PHA fermentation medium. For characterization of the cloned promoters by a reporter gene assay, the commonly used LB medium for the various reporter gene assays was also selected for this study 11,13 . The well-characterized promoters in LB medium may also have the potential to be applied for the synthesis of other products in P. mendocina. However, the transcriptional levels of the 10 candidate promoters obtained by RNA-seq analysis may not always be consistent with the transcriptional levels measured by qPCR due to the different culture conditions. For example, the promoters P17, P18 and P29 had high RPKM values in the RNA-seq analysis, but they exhibited lower transcriptional levels in the reporter gene assay than lac promoter at any growth phase. In the future, more RNA-seq data based on different culture conditions should be overall considered to select the candidate promoters. Then, through characterization of the putative promoters by a reporter gene assay in LB medium, the screened strong endogenous promoters may have a wide-range application for various products in P. mendocina.

Characterization of the cloned promoters via GFP fluorescence measurement

To further determine the expression levels of the selected endogenous promoters, relative fluorescence intensities were measured at three different growth phases. As shown in Fig. 3, the five strong promoters P4, P6, P9, P16 and P25 characterized by qPCR had also higher relative fluorescence intensities than lac promoter at any growth phase. P4 had the strongest relative fluorescence intensity among the 10 selected promoters, which showed a nearly 32-fold enhancement compared with lac promoter at the stationary phase. For each of the above five strong promoters, significant difference in the intensity of GFP fluorescence was observed at different growth stages, which was in agreement with the previous results on the unstable transcriptional levels of gfp measured by qPCR (Fig. S3). All of the results suggest that the expression levels of the five strong promoters might not be constant over the entire growth cycle (Fig. 3), The orders of promoter strength reflected by the real-time qPCR and GFP reporter were identical to the result obtained by RNA-seq (RPKM value), which demonstrated that the results of transcriptome sequencing analysis and functional validation experiments were highly consistent.

Characterization of the chosen promoters and lac promoter via GFP fluorescence intensity measurements. Expression of gfp gene under different promoters in P. mendocina NKU was quantified at different growth phases. The background expression was subtracted, and the relative fluorescence intensity was calculated by normalization against per OD600 of whole cells. Data represent the mean values ± standard deviations of triplicate measurements from three independent experiments. A Student’s t-test was performed between lac promoter and chosen promoters. * and ** indicate P < 0.05 and P < 0.01, respectively.

Interestingly, the relative transcriptional level of P25 was higher than that of P6, P9 and P16 (Fig. 2), but the relative fluorescence intensity of P25 was lower than that of P6, P9 and P16 (Fig. 3). The relative fluorescence intensities of the remaining promoters P17, P18, P20, P23 and P29 were lower than that of lac promoter at any growth phase, although P20 and P23 showed higher transcriptional levels than lac promoter. The observations suggest that the high transcriptional level of a gene might not necessarily lead to the high-level synthesis of this protein encoded by the gene. To maintain the consistency of translation initiation efficiency, in this study, the same RBS was introduced into upstream of reporter gene gfp. Among the reporter gene vectors, the distance between the predicted promoter sequences and RBS was different from each other, which may affect the efficiency of mRNA translation, possibly leading to the discrepancy between the transcriptional level and fluorescence intensity. For example, the distance between the predicted promoter sequences and RBS for P25 were longer than that for P6, P9 and P16. This may be the reason why P25 had higher transcriptional level, but lower fluorescence intensity than those of P6, P9 and P16. In addition, the differences in the spacer sequences between promoter and RBS may be the second reason for the different trends between the transcriptional level and fluorescence intensity.

When observed by confocal microscopy, cells expressing gfp under the control of P4, P6, P9, P16 and P25 produced more bright green fluorescence than the control cells with gfp expression under the control of lac. Cells produced weak green fluorescence when gfp expression was driven by P23 and P20. However, green fluorescence was not observed on the cells when expression of gfp was under the control of P17, P18 and P29 (Fig. 4). The results from confocal microscope matched well with that from GFP fluorescence intensity measurement.

Characterization of the chosen promoters and lac promoter via confocal microscope. (A) Green fluorescence within the cell. (B) Outline of cell membrane by stain with FM4-64/L. (C) A and B merged together. All the images were taken at the same exposure condition.

In a previous study, a set of synthetic promoters, which is capable of stable and constitutive expression of downstream genes, was applied for calibrated heterologous gene expression in P. putida KT2440 using a mini-Tn7 delivery transposon vector that inserts the promoters into the genome of P. putida 28 . In another study, different inducible promoters were characterized by the construction of ProUSER-reporter vectors for use in P. putida KT2440, and the production of p-coumaric acid in P. putida KT2440 was enhanced by the use of selected inducible promoters for the optimization of pathway expression 29 . In this work, the five endogenous strong promoters P4, P6, P9, P16 and P25 were identified from P. mendocina NK-01 using a pipeline consisting of RNA-seq analysis and transcriptional level and fluorescence intensity measurements of a reporter gene gfp. So far, very little is known about the screening of strong promoters from the genus Pseudomonas using a RNA-seq-based strategy.

Enhanced production of PHA by overexpressing phaC using the strong promoters in P. mendocina NKU

The mcl-PHA synthetic operon of P. mendocina NK-01 had been expounded in the earlier research. Our study has shown that PhaC1 is the main contributor to mcl-PHA synthesis in P. mendocina NK-01 23 . Consequently, overexpressing PHA synthase genes, especially the phaC1 gene, may have a positive influence on mcl-PHA accumulation. The Standard European Vector Architecture Database (SEVA) has developed a series of plasmid vectors for metabolic engineering and synthetic biology in Pseudomonas and other gram-negative bacteria 30,31 . However, plasmid expression systems tend to be a burden on the bacteria, especially when multiple genes are needed to be co-expressed in a bacterium. In this work, the 3 endogenous strong promoters P4, P6 and P16 were selected for overexpressing PHA synthase genes by unmarked insertion of promoters upstream of the phaC1 gene in the genome of P. mendocina NKU. This process did not leave any redundant sequences in the genome except the inserted promoter sequences (Fig. 5). This scarless genome editing strategy may confer some advantages over plasmid-borne overexpression of phaC genes.

The construction schematic diagram for inserting the promoters into upstream of phaC1 gene and for knockout of phaZ in the genome of P. mendocina NKU.

Through chromosomal insertion of the 3 endogenous strong promoters P4, P6 and P16, the transcriptional levels of phaC1 and phaC2 in the recombinant strains NKU-4C1, NKU-6C1 and NKU-16C1 were all improved compared with strain NKU. In particular, phaC1 showed a more obvious improvement than phaC2 (Fig. 6). This may be due to the fact that phaC1 is closer to the inserted promoters than phaC2.

qPCR analysis and PHA fermentation results for the strains NKU-4C1, NKU-6C1, NKU-16C1 and NKU. Transcriptional levels of phaC1 (a), phaC2 (b) and phaZ (c) for the different strains. (d) Cell dry weight (CDW) and PHA production for the strains. Samples for qPCR were taken at 36 h of PHA fermentation. The transcriptional level for strain NKU was set as 1. wt% was defined as the ratio of PHA to CDW. Data represent the mean values ± standard deviations of triplicate measurements from three independent experiments. A Student’s t-test was performed between NKU and the mutants. * and ** indicate P < 0.05 and P < 0.01, respectively.

Moreover, the phaZ located between phaC1 and phaC2 had high transcriptional levels in the recombinant strains. Especially for NKU-6C1 and NKU-16C1, the transcriptional levels of phaZ were improved even more than phaC1, even though phaZ was far from the promoter in the gene cluster when compared to phaC1. This could because of that tight regulatory coupling between PHA polymerase activity and depolymerase activity may exist in this strain. It had been reported that single overexpression of PhaC may lead to an increase in the expression of PhaZ 32 . So phaZ showed a higher transcriptional level than phaC1, when PhaC and PhaZ were overexpressed simultaneously in a gene cluster. The PHA fermentation results showed that the PHA titers of NKU-4C1, NKU-6C1 and NKU-16C1 were all reduced, especially in NKU-6C1 (Fig. 6). These observations suggest that the overexpression of phaZ may lead to excessive synthesis of PHA depolymerase, and that the intracellularly accumulated PHA may be degraded by the depolymerase. The regulatory roles of PHA depolymerase in the synthesis of PHA were investigated previously by other researchers. For example, overexpression of PhaC2 alone in P. putida strain U was unable to accumulate higher amounts of PHA than in the wild-type strain, as a result of elevated PHA depolymerization in the late stage of PHA synthesis. A phaZ-inactive mutant of P. putida strain U, however, accumulated higher levels of PHA than the parental strain 32 . The mcl-PHA content of a phaZ knockout mutant of P. putida KT2442 (86 wt%, the ratio of PHA to CDW) was higher than that of wild-type strain (66 wt%) when using sodium octanoate as the carbon source 33 . However, the elimination of PHA depolymerase activity in P. putida KT2440 had little impact on the overall yield of PHA 34 . Both a phaZ-deficient mutant of P. oleovorans GPo1 35 and two transposon-disrupted phaZ mutants of P. resinovorans 36 did not show any substantial increase in PHA titer under various PHA synthesis conditions.

In this work, we attempt to improve the yield of PHA by the construction of phaZ knockout mutants. However, the PHA titer of strain NKU-phaZ was decreased by 4 wt% compared with strain NKU from 21 to 17 wt% (Fig. 7), indicating that knockout of phaZ cannot improve the yield and molecular weight of mcl-PHA in P. mendocina NK-01. Surprisingly, PHA synthesized by all phaZ knockout mutants had lower molecular weights than PHA synthesized by the parent strain NKU, with an exception of NKU-phaZ-6C1 (Table S2). mcl-PHAs synthesized by P. mendocina NKU and its mutant strains were mainly composed of three different monomers, i.e., 3-hydroxyoctanoate, 3-hydroxydecanoate and 3-hydroxydodecanoate, as shown by GC-MS analysis (Figs S4, S5). The monomer composition ratios of the mcl-PHAs had not obvious changes for the mutants compared with NKU (Table S3).

qPCR analysis and PHA fermentation results for the strains NKU-∆phaZ-4C1, NKU-∆phaZ-6C1, NKU-∆phaZ-16C1 and NKU-∆phaZ. Transcriptional levels of phaC1 (a), phaC2 (b) and phaZ (c) for the different strains. (d) CDW and PHA production for the strains. Samples for qPCR were taken at 36 h of PHA fermentation. The transcriptional level for strain NKU-∆phaZ was set as 1. Data represent the mean values ± standard deviations of triplicate measurements from three independent experiments. A Student’s t-test was performed between NKU-∆phaZ and other mutants. * and ** indicate P < 0.05 and P < 0.01, respectively.

The relative transcriptional values of phaC1 and phaC2 in strain NKU-phaZ-4C1, NKU-phaZ-6C1 and NKU-phaZ-16C1 were all improved compared with NKU-phaZ, (Fig. 7). Interestingly, the relative transcriptional values of phaC1 and phaC2 in strain NKU-phaZ-16C1 were the lowest among the above three strains, while the PHA titer of strain NKU-phaZ-16C1 was the highest among the above three strains. The PHA titer of strain NKU-phaZ-4C1 was similar to that of strain NKU-phaZ. Compared with strain NKU-phaZ, the PHA titer of strain NKU-phaZ-6C1 was reduced by 7%, and the PHA titer for strain NKU-phaZ-16C1 were improved by 6% to 23 wt% (Fig. 7). These results indicated that the expression level of phaC was not positively related to the PHA titer in strain NK-01. In future studies, the optimal expression of phaC may be required for obtaining the highest PHA yield in strain NK-01. It should be noted that the native RBS sequence of the phaC gene was unchanged when the strong endogenous promoters were inserted into upstream of the phaC operon in the genome of P. mendocina. In P. mendocina, the native RBS sequence may be optimal for the translational initiation of the phaC operon. RBS can serve as an important regulatory element for translational initiation and thus obviously affect the gene expression level 37,38 . Only using strong promoters may not obtain the optimal expression levels. For this study, the optimization of the RBS sequence coupled with the screening of endogenous strong promoters may be required for the optimal PHA synthase gene expression.

The relative transcriptional values of phaC1 and phaC2 for the different mutant strains at 12 h and 24 h of mcl-PHA fermentation were also measured, respectivly. As expected, the strains NKU-phaZ-4C1, NKU-phaZ-6C1 and NKU-phaZ-16C1 showed higher transcriptional levels for phaC1 and phaC2 than NKU-phaZ at 12 h, however, no significant differences were observed among the above three strains (Fig. S6). At 24 h, the relative transcriptional values of phaC1 for the above three strains were also improved compared with NKU-phaZ, and NKU-phaZ-4C1 had the highest transcriptional level among the three strains (Fig. S6). Surprisingly, the transcriptional levels for phaC2 at 24 h had not obvious increase for NKU-phaZ-4C1, NKU-phaZ-6C1 and NKU-phaZ-16C1 compared with NKU-phaZ (Fig. S6). The strain NKU-phaZ-16C1 showed the lowest transcriptional levels for phaC1 and phaC2 in any timepoints (Fig. 7 and Fig. S6). These results indicated that the transcriptional levels of phaC1 and phaC2 for the mutant strains were not constant during the PHA fermentation. The changes in the transcriptional levels of the PHA synthase genes over the PHA fermentation period may contribute to lower increase in the PHA yield.

Since a complex metabolic pathway is involved in the synthesis of PHA from glucose, there are many important factors to influence the efficiency of PHA synthesis, including the Entner-Doudoroff pathway, the flux of acetyl-CoA, the fatty acid de novo synthesis pathway, and the availability of the PHA synthesis 39,40,41 . Modification of a few factors may not have an obvious influence for the improvement of PHA synthesis.

In this work, all phaZ knockout mutants showed a decrease in the relative transcriptional levels of phaC1 and phaC2 compared with their corresponding strains without deletion of phaZ. Compared with strain NKU-phaZ, the reduction in the PHA titer was observed with strain NKU-phaZ-4C1 and NKU-phaZ-6C1, while the PHA titer was improved for strain NKU-phaZ-16C1 (Fig. 7). These results suggest that PhaZ is not only involved in PHA degradation but also acts as an important role in PHA synthesis in P. mendocina NK-01. A previous study has shown that PhaZ may play a crucial role in the turnover of mcl-PHA under starvation conditions in P. putida KT2442 42 . Rational tuning of the transcriptional activity of PHA synthase and depolymerase would be a feasible approach for the optimization of PHA production in strain NK-01. Therefore, we believe that the screened endogenous strong promoters have the potential to be applied for overexpression of PHA synthesis pathway genes to improve the production of PHA in P. mendocina NK-01.

Promoter engineering such as the screening of strong promoters has been widely applied for metabolic pathway engineering to improve the yield of many industrial products. However, in many cases, the exogenous promoters may not be compatible with the native gene expression systems in P. mendocina NK-01. A previous study in our lab showed that the PHA yield had an obvious decrease after the overexpression of PHA synthase genes using an exogenous strong promoter J23119 in P. putida KT2440 (unpublished data). This also shown it’s not that the more of PhaC expression was, the higher of mcl-PHA yield could get. In this study, we didn’t select a very strong exogenous promoter as the reference and the commonly used lac promoter 43,44 has been used as the control in the transcriptional activity assays to screen appropriate endogenous strong promoters. Compared with the lac promoter, the screened endogenous promoters P4, P6 and P16 showed higher transcriptional activity and fluorescence intensity. Therefore, we tested the ability of the screened endogenous promoters to improve the production of mcl-PHA by overexpressing PHA synthase in P. mendocina NK-01. Future work is needed to screen more suitable promoters and optimize the PHA biosynthetic pathway to further improve the mcl-PHA production, not only via overexpression of PHA synthase genes. And the screened endogenous promoters can also be applied to enhance the biosynthesis of AO which is another product synthesized by NK-01 from glucose. The use of endogenous promoters may be a feasible method for the optimization of the expression of the synthetic pathway genes, and this strategy could be potentially utilized for enhanced production of other valuable bio-based products.


Contents

The two most commonly used inducible expression systems for research of eukaryote cell biology are named Tet-Off and Tet-On. [3] The Tet-Off system for controlling expression of genes of interest in mammalian cells was developed by Professors Hermann Bujard and Manfred Gossen at the University of Heidelberg and first published in 1992. [4]

The Tet-Off system makes use of the tetracycline transactivator (tTA) protein, which is created by fusing one protein, TetR (tetracycline repressor), found in Escherichia coli bacteria, with the activation domain of another protein, VP16, found in the Herpes Simplex Virus. [5]

The resulting tTA protein is able to bind to DNA at specific TetO operator sequences. In most Tet-Off systems, several repeats of such TetO sequences are placed upstream of a minimal promoter such as the CMV promoter. The entirety of several TetO sequences with a minimal promoter is called a tetracycline response element (TRE), because it responds to binding of the tetracycline transactivator protein tTA by increased expression of the gene or genes downstream of its promoter.

In a Tet-Off system, expression of TRE-controlled genes can be repressed by tetracycline and its derivatives. They bind tTA and render it incapable of binding to TRE sequences, thereby preventing transactivation of TRE-controlled genes.

A Tet-On system works similarly, but in the opposite fashion. While in a Tet-Off system, tTA is capable of binding the operator only if not bound to tetracycline or one of its derivatives, such as doxycycline, in a Tet-On system, the rtTA protein is capable of binding the operator only if bound by a tetracycline. Thus the introduction of doxycycline to the system initiates the transcription of the genetic product. The Tet-On system is sometimes preferred over Tet-Off for its faster responsiveness.

Tet-Off expression systems are also used in generating transgenic mice which conditionally express gene of interest.

The Tet-On Advanced transactivator (also known as rtTA2 S -M2) is an alternative version of Tet-On that shows reduced basal expression, and functions at a 10-fold lower Dox concentration than Tet-Off. In addition, its expression is considered to be more stable in eukaryotic cells due to being human codon optimized and utilizing 3 minimal transcriptional activation domains. It was discovered in 2000 as one of two improved mutants by H. Bujard and his colleagues after random mutagenesis of the Tet Repressor part of the transactivator gene. [6] Tet-On 3G (also known as rtTA-V10 [7] ) is similar to Tet-On Advanced but was derived from rtTA2 S -S2 rather than rtTA2 S -M2. It is also human codon optimized and composed of 3 minimal VP16 activation domains. However, the Tet-On 3G protein has 5 amino acid differences compared to Tet-On Advanced which appear to increase its sensitivity to Dox even further. Tet-On 3G is sensitive to 100-fold less Dox and is 7-fold more active than the original Tet-On. [8]

Other systems such as the T-REx system by Life Technologies work in a different fashion. [9] The gene of interest is flanked by an upstream CMV promoter and two TetO2 sites. Expression of the gene of interest is repressed by the high affinity binding of TetR homodimers to each TetO2 sequences in the absence of tetracycline. Introduction of tetracycline results in binding of one tetracycline on each TetR homodimer followed by release of TetO2 by the TetR homodimers. Unbinding of TetR homodimers and TetO2 result in derepression of the gene of interest.

A modified version of T-REx is the Linearizer synthetic biological circuit, optimized for gene expression tuning in eukaryotic (budding yeast, human, etc) cells. By incorporating TetO2 sites into the promoter driving TetR expression, it creates negative feedback, which ensures homogeneous expression (low noise) and a linear dose-response to tetracycline analogs. [10]

In the most commonly used plasmids, the tetracycline response element consists of 7 repeats of the 19bp bacterial TetO sequence ( TCCCTATCAGTGATAGAGA ) separated by spacer sequences (for example: ACGATGTCGAGTTTAC ). It is the TetO that is recognized and bound by the TetR portion of Tet-On or Tet-Off. The TRE is usually placed upstream of a minimal promoter that has very low basal expression in the absence of bound Tet-Off (or Tet-On).

The Tet system has advantages over Cre, FRT, and ER (estrogen receptor) conditional gene expression systems. In the Cre and FRT systems, activation or knockout of the gene is irreversible once recombination is accomplished, whereas, in Tet and ER systems, it is reversible. The Tet system has very tight control on expression, whereas ER system is somewhat leaky. [11] However, the Tet system, which depends on transcription and subsequent translation of a target gene, is not as fast-acting as the ER system, which stabilizes the already-expressed target protein upon hormone administration. Also, since the 19bp tet-o sequence is naturally absent from mammalian cells, pleiotropy is thought to be minimized compared to hormonal methods of control. When using the Tet system in cell culture, it is important to confirm that each batch of fetal bovine serum is tested to confirm that contaminating tetracyclines are absent or are too low to interfere with inducibility.

The mechanism of action for the antibacterial effect of tetracyclines relies on disrupting protein translation in bacteria, thereby damaging the ability of microbes to grow and repair however protein translation is also disrupted in eukaryotic mitochondria leading to effects that may confound experimental results. [12] [13]


Methods

Chick embryos

Fertilized chick eggs from a commercial source (JA57 strain, Dangers, France) were incubated at 38.5 °C. Embryos were staged according to days in ovo. For early stages, the following day numbers and HH (Hamburger and Hamilton) stages [56] are equivalent: E2/HH13, E2.5/HH15 and correspond to 20 and 25 somite stages, respectively.

Establishment of recombinant vectors

The pT2AL-MLC-Tomato-T2A-GFP plasmid was obtained as following: The Myr-TdTomato-T2A sequence was amplified by PCR from the plasmid pCS2-TdTomato-2A-GFP [52]. To facilitate subsequent cloning, one XhoI site was added to the forward primer and one BstBI site was added to the reverse primer. The purified PCR product was then inserted into pCRII-TOPO (Invitrogen) and a clone with Tomato downstream of SP6 promoter was selected, giving rise to a plasmid named TOPO/Tomato. H2B-GFP was amplified by PCR from the plasmid pCS2-TdTomato-2A-GFP [52]. A BstbI site was added to the forward primer and one PmlI site and one ClaI site were added to the reverse primer. The purified PCR product was then inserted into pCRII-TOPO (Invitrogen) and a clone with GFP downstream of SP6 promoter was selected, resulting in a plasmid called TOPO/GFP. Next, both TOPO/Tomato and TOPO/GFP were digested with BstbI and NotI. The T2A sequence was then inserted into TOPO/Tomato using the T4 DNA ligase (New England Biolabs) to generate a plasmid named TOPO/Tomato-T2A-GFP. The Tomato-T2A-GFP cassette was then excised from TOPO Tomato-T2A-GFP using EcoRV and XhoI and cloned into the pT2AL200R150G [57] previously digested with ClaI (blunt-ended using Fermentas T4 DNA polymerase) and XhoI. The resulting plasmid was named pT2AL-Tomato-T2A-GFP. The Myosin Light Chain (MLC) mouse promoter was removed from the pT2K-MLC-Fgf4 plasmid (previously described in [44]) using NcoI and XhoI. Both extremities were then blunt-ended using T4 DNA polymerase (Fermentas). The MLC promoter was next blunt ligated to TOPO GFP previously digested with XbaI made blunt. A clone with the MLC promoter inserted with ApaI in 5’ and XhoI in 3’ was selected resulting in a plasmid called TOPO/GFP/MLC. Both TOPO/GFP/MLC and pT2AL-Tomato-T2A-GFP were digested with ApaI and XhoI. MLC was inserted into pT2AL-Tomato-T2A-GFP to obtain pT2AL-MLC-Tomato-T2A-GFP.

The pT2AL-CMV/βactin-Tomato-T2A-GFP plasmid was obtained as followed: The pT2AL-MLC-Tomato-T2A-GFP plasmid was digested with ApaI (blunt-ended using Fermentas T4 DNA polymerase) and SphI to remove the MLC promoter. The MLC promoter was then replaced by the CMV-βactin promoter (the chick βactin promoter downstream of a CMV enhancer), which was excised from the CMV-βactin-EGFP [35] using SalI (blunt-ended) and SphI to generate the pT2AL-CMV-Tomato-T2A-GFP plasmid.

The pT2AL-p57/βactin -Tomato-T2A-GFP plasmid was obtained as followed: The p57MRE regulatory sequence was excised from pSK-p57MRE plasmid [11] by digestion with Spe1 and SacII. The CMV enhancer of the pT2AL-CMV/βactin-Tomato-T2A-GFP plasmid was excised by Acc1 and SnaBI and replaced with the p57MRE using blunt ligation with the Rapid DNA Ligation Kit (Roche). The generated plasmid was named the pT2AL-p57/βactin-Tomato-T2A-GFP.

Electroporation

Limb somite electroporation was performed as previously described [35]. The DNA solution was systematically composed of the Tol2 stable vectors and the transient transposase vector CMV/βactin-T2TP, which allows the stable integration into the chick genome. The concentration of the different vectors was between 1.5 and 2 μg/μL and of 1/3 for the CMV/βactin-T2TP. DNA was prepared in solution containing carboxymethyl cellulose 0,17 %, fast green 1 %, MgCl2 1 mM and PBS 1X in water.

Lateral plate electroporation was performed as followed: Stage HH13–15 (E2) chick embryos were windowed following standard techniques in preparation for electroporation [58]. PBS without Ca 2+ /Mg 2+ was applied to the embryo. A capillary was backfilled with DNA solution, which was injected under 200 Pa pressure (injection duration 0.1–0.5 s and compensatory pressure 15–25 Pa) (Femtojet, Eppendorf) into the embryonic coelom, to fill completely the anterior to posterior extent of the forelimb territory. The negative electrode (0.8 mm diameter tungsten rod with a 4-mm length and 2-mm exposed surface) was inserted into the yolk and positioned beneath the forelimb field, approximately 2 mm below the embryo. A 0.8 mm diameter platinum rod with a 1-mm exposed tip served as the positive electrode and was positioned above the forelimb field with an approximate distance of 3 mm. A wave pulse train consisting of 50 V, five pulses, 20 ms duration with a 200 ms interpulse interval was delivered via TSS20 electroporator and EP21 current amplifier (Intracel). Embryos were returned to 37.5 °C for the remaining incubation period. DNA solution was composed of pT2AL-CMV/βactin-Tomato-T2A-GFP (1-3 μg/μL) and CMV/βactin-T2TP at a molar ratio of 1:5–1:10, diluted in a mix containing PBS without Ca 2+ /Mg 2+ and Fast Green 0.005 %. This ratio resulted in persistent gene expression in the embryonic limbs during foetal development.

Immunohistochemistry

Experimental forelimbs were fixed in paraformaldehyde 4 % overnight at 4 °C and processed for cryostat sections (12 μm). Immunohistochemistry was performed as previously described [59]. The monoclonal antibodies MF20 that recognizes sarcomeric myosin heavy chains and Pax7 that recognizes muscle progenitors, developed by D.A. Fischman and A. Kawakami, respectively, were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology Iowa City, IA 52242. After overnight incubation with the primary antibody at 4 °C, biotinylated secondary antibodies (Anti-Mouse IgG2b from Southern Biotech Anti-Mouse IgG1 from Jackson ImmunoResearch laboratories) were applied for 1 h at room temperature, followed by a 45 min incubation with Cy5-Streptavidin (Invitrogen). Hoechst (Molecular Probes) staining was performed with a dilution of 1/20000 in PBS 1X for 10 min at room temperature.

In situ hybridization

In situ hybridization experiments were performed for GFP and Scx probes, as previously described [35].

Image capturing

Images of the wholemount electroporated limbs were acquired with a Leica stereo-macroscope equipped with a Leica DFC300 camera. After immunohistochemistry, sectioned samples images were captured using a Nikon epifluorescence microscope, a Leica DMI600B inverted microscope or a Leica SP5 confocal system.


MATERIALS AND METHODS

ASC Isolation and Clonal Culture

Stromal vascular cells with a CD34 + CD105 + CD45 − CD31 − phenotype (ASCs) were isolated from human adipose tissue (Boquest et al., 2005). In short, tissue was obtained by liposuction from the hip and thigh regions of healthy women. After washing in Hank’s balanced salt solution (HBSS), the tissue was digested for 2 h at 37°C in HBSS with collagenase and DNase I. Adipocytes were separated from stromal vascular cells after sedimentation at 400 × g for 10 min and removed by aspiration. Erythrocytes were removed by resuspending stromal vascular cell pellets in lysis buffer (2.06 mg/ml Tris base, pH 7.2, and 7.49 mg/ml NH4Cl) for 10 min. After centrifugation, pellets were resuspended in HBSS containing 2% fetal bovine serum (FBS) (Sigma-Aldrich. St. Louis, MO) and passed through a 100-μm sieve and a 40-μm sieve. CD45 + cells were removed with paramagnetic beads conjugated to mouse anti-human CD45 monoclonal antibodies (Miltenyi Biotech, Bergish Gladbach, Germany) using a superMACS magnet (Miltenyi Biotech). Remaining CD45 − cells were incubated with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD31 antibodies (Serotec, Oxford, United Kingdom) at a concentration of 10 μl of antibody per 10 6 cells for 15 min at 4°C. Cells were washed and incubated with anti-FITC microbeads (Miltenyi Biotech) for 15 min at 4°C. CD31 − and CD31 + cells were separated using an LS column (Miltenyi Biotech). CD31 − cells were reexposed to a new LS column to eliminate any leftover contaminating CD31 + cells. Flow cytometry analysis of each cell subset from each donor indicated that purity was >98% (our unpublished data) (Boquest et al., 2005). Aliquots of each cell subset were immediately snap-frozen in liquid nitrogen for DNA and RNA isolations, or they were cultured.

CD31 − clonal cell lines were generated by culturing single CD31 − cells in each well of 48-well plates in DMEM/F-12 medium containing 50% FBS and antibiotics. After ∼16 h, the medium was replaced by DMEM/F-12 with 20% FBS. After ∼1 wk, colonies containing >10 cells were passaged by trypsinization and expanded. Only clonal lines that could be easily expanded were used in this study. Clones A1 and A2, and clones B1, B2, and B3 examined in this study were from two different female donors (age 27 and 39, respectively).

Adipogenic Differentiation

Clonal ASC lines generated from individual CD31 − cells at passage 4 were cultured to confluence before differentiation. For adipogenic differentiation (Zuk et al., 2001), cells cultured in DMEM/F-12 with 10% FBS were stimulated for 3 wk with 0.5 mM 1-methyl-3 isobutylxanthine, 1 μM dexamethasone, 10 μg/ml insulin (Novo Nordisk, Copenhagen, Denmark), and 200 μM indomethacin (Dumex-Alpharma, Copenhagen, Denmark). To visualize lipid droplets, formalin-fixed cells were washed in 50% isopropanol and stained with Oil Red-O.

Gene Loci and Regions Analyzed by Bisulfite Sequencing

Supplemental Figure S1 illustrates the promoter regions of the genes analyzed by bisulfite sequencing in this study. We examined four adipogenic genes, including leptin (LEP) (Mason et al., 1998 Reseland et al., 2001), peroxisome proliferator-activated receptor gamma 2 (PPARG2) (Fajas et al., 1997), fatty acid-binding protein 4 (FABP4) (Ross et al., 1990 Graves et al., 1992), and lipoprotein lipase (LPL) (Bey et al., 1998 Merkel et al., 2002). We also examined genes unrelated to adipogenesis, such as myogenin (MYOG), a basic helix-loop-helix transcription factor required for myocyte differentiation (Massari and Murre, 2000) the endothelial marker gene CD31/PCAM-1 (Cao et al., 2002 Chi et al., 2003) and the constitutively expressed housekeeping gene GAPDH. The LEP promoter region analyzed was from nucleotides 2719–2937 (GenBank accession no. U43589) and spanned 27 potentially methylated cytosines in CpG dinucleotides starting 42 base pairs upstream of the ATG translational start site. The LEP proximal promoter activity is known to be regulated by DNA methylation (Melzner et al., 2002). The PPARG2 promoter region (Fajas et al., 1997) spanned nucleotides 108–587 (GenBank accession no. AB005520) and included 6 CpGs starting 264 base pairs upstream of the ATG. The FABP4 (GenBank accession no. NM_001442) promoter region examined was identified using ENSEMBL and encompassed four CpGs starting 130 base pairs upstream of the ATG. The LPL promoter region spanned bases 1321–1777 (GenBank accession no. X68111) and included 11 CpGs starting 134 base pairs upstream of the ATG. The MYOG region analyzed spanned nucleotides 1268–1484 (GenBank accession no. X62155) and included 16 CpGs starting 87 base pairs downstream of the ATG. The CD31 promoter region examined included nucleotides 1095–1480 (GenBank accession no. X96848) and included 18 CpGs ranging from nucleotide −352 to +34 relative to the ATG. The GAPDH promoter region spanned bases 1121–1337 (GenBank accession no. J04038) and encompassed 28 CpGs 116 base pairs upstream of the ATG.

Bisulfite Sequencing

DNA was purified either using the GenElute Mammalian Genomic DNA Miniprep kit (Sigma-Aldrich), or for most samples, by phenol-chloroform-isoamyl alcohol extraction. In the latter case, cells were first lysed for 10 min in lysis buffer (10 mM Tris-HCl, pH 8, 100 mM EDTA, and 0.5% SDS) and digested with 0.1 mg/ml proteinase K overnight. Bisulfite conversion (Warnecke et al., 2002) was performed using the MethylEasy DNA bisulfite modification kit (Human Genetic Signatures, Sydney, Australia). Converted DNA was used fresh or stored at −20°C. Converted DNA was amplified by PCR using primer sets purchased from Human Genetic Signatures for the LEP, MYOG, CD31 and GAPDH genes. These primers sets are commercially available (www.geneticsignatures.com). We also designed primers using the Methprimer software (www.urogene.org/methprimer/index1.html) for the PPARG2, FABP4, and LPL genes (Table 1). For PPARG2, FABP4, and LPL, PCR conditions were 95°C for 7 min and 40 cycles of 95°C 1 min, 54°C 2 min and 72°C 2 min, followed by 10 min at 72°C. For LEP, MYOG, CD31, and GAPDH, nested PCRs were performed, each as follows: 95°C for 3 min and 30 cycles of 95°C for 1 min, 50°C for 2 min, and 72°C for 2 min, followed by 10 min at 72°C. PCR products were directly sequenced or cloned into bacteria using the TOPO TA cloning kit (Invitrogen, Oslo, Norway). Clones were sequenced using commercial services from MWG Biotech (Ebersberg, Germany).

Table 1. Bisulfite sequencing primers used in this study

a Purchased from Human Genetic Signatures.

Real-Time Reverse Transcription (RT)-PCR

RT-PCR was carried from 500 ng of total RNA using the Iscript cDNA synthesis kit (Bio-Rad, Hercules, CA). Quantitative (Q)RT-PCR reactions were performed in triplicates on a MyiQ real-time PCR Detection System using IQ SYBR Green (Bio-Rad). Most samples were analyzed in duplicates from two separate cDNA preparations. Primers used are listed in Table 2. SYBR Green PCR conditions were 95°C for 4.5 min and 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, using GAPDH as a normalization control. mRNA levels were calculated as described previously (Pfaffl, 2001).

Table 2. Real-time RT-PCR primers used in this study


Expression Vectors: Types & Characteristics

The expression vectors are vectors which act as vehicles for DNA insert and also allow the DNA insert to be expressed efficiently. These may be plasmids or viruses. The expression vectors are also known as expression constructs.

The expression vectors are genetically engineered for the introduction of genes into the target cells. In addition to the gene of interest, these expression constructs also contain regulatory elements like enhancers and promoters so that efficient transcription of the gene of interest occurs.

The simplest expression constructs are also known as transcription vectors only because they allow transcription of the cloned foreign gene and not its translation. The vectors which facilitate both transcription and translation of the cloned foreign gene are known as protein expression vectors. These protein expression constructs also lead to the production of recombinant protein.

Now, for transcription and translation, a promoter and a termination sequence are a must. Transcription initiates at the promoter and ends at the termination site. The promoters of expression vectors must have on/off switches. These switches help in the regulation of production of the gene product. Excessive amounts of product of the gene of interest can be toxic for the cell. A common promoter utilized in the expression constructs is the mutant version of the lac promoter, lacUV. The lacUV promoter initiates a high level of transcription under induced conditions. Moreover, in some expression vectors, a ribosomal binding site is present upstream to the start codon. The ribosomal binding site facilitates the efficient translation of the cloned foreign gene.

Expression vectors are used extensively in molecular biology in techniques like site-directed mutagenesis.

How do Expression Vectors work?

  • Once the expression construct is inside the host cell, the protein encoded by the gene of interest is produced by the transcription. Thereafter, it utilizes the translation machinery and ribosomal complexes of the host organism.
  • Frequently, the plasmid is genetically engineered to harbor regulatory elements like enhancers and promoters. These regulator sequences aid in efficient transcription of the gene of interest.
  • Expression vectors are extensively used as tools which help in the production of mRNAs and, in turn, stable proteins. They are of much interest in biotechnology and molecular biology for the production of proteins like insulin. Insulin is the chief ingredient in the treatment of the complex disease, Diabetes.
  • When the protein product is expressed, it is to be then purified. The purification of a protein poses a challenge since the protein of interest, whose gene is carried on the expression vector, is to be purified independently of the proteins of the host organism. To make the process of purification simpler, the gene of interest carried on the expression vector should always have a ‘tag’. This tag can be any marker peptide or histidine (His tag).
  • Expression vectors are considerably exploited in techniques like site-directed mutagenesis. Cloning vectors introduce the gene of interest into a plasmid which in turn replicates in bacteria. These cloning vectors need not necessarily result in the expression of a protein.

Therefore, expression vectors must have the following expression signals:

  • Strong promoter,
  • Strong termination codon,
  • Adjustment of distance between the promoter and cloned gene,
  • Inserted transcription termination sequence, and
  • Portable translation initiation sequence.

Promoter

  • A promoter ensures a reliable transcription of the gene of interest. Also, strong promoters are also necessary for an efficient mRNA synthesis with RNA polymerase.
  • Regulation of the promoter is another critical aspect which should always be kept in mind while constructing an expression vector.
  • The strongest promoters are those found in bacteriophages T5 and T7.

In E. coli, the promoter is regulated in two ways:

Induction : the addition of chemical switches on the transcription of the gene.

Repression : addition of chemical switches off the transcription of the gene.

The most commonly used promoters in E. coli expression system are:

  • It regulates the transcription of the lac Z gene. The lac Z gene is responsible for the production of β- galactosidase.
  • The lac Z gene can be induced by IPTG, isopropylthiogalactosidase.
  • The lac promoter sequences can be fused to the target gene. It will, then, result in lactose- dependent expression of the target gene.
  • Nevertheless, the lac promoter has its drawbacks. It is quite weak and cannot be utilized for the high levels of production of the desired protein. In addition to this, the lac genes carry out the basal level of transcription even in the absence of induction (inducer molecule).
  • It is responsible for the regulation of a cluster of genes which are involved in tryptophan biosynthesis.
  • Tryptophan acts as its repressor molecule, and it is induced by 3-β-indoleacrylic acid.
  • It is formed by hybridization of the lac and trp promoter. However, it is stronger than either of them.
  • The tac promoter is induced by IPTG, isopropylthiogalactosidase.
  • It is a strong promoter and is responsible for transcription of λDNA in E. coli
  • The product of λcI gene acts as its repressor. It is called λ repressor.
  • The expression construct with the λPL promoter is used in combination with the E. coli mutant host. It is responsible for the production of a temperature sensitive form of λ repressor.
  • At low temperatures, the repressor protein represses the transcription whereas the transcription of the cloned gene occurs at high temperatures because the repressor is inactivated at high temperature.
  • For the expression of proteins in mammalian cells, the promoter must be located upstream of the cloned cDNA for its efficient transcription.
  • In most of the cases, viral promoters are employed only because they are reliable for a strong constitutive expression.
  • The widely used promoters are CMV promoter (derived from cytomegalovirus) and the SV40 promoter (derived from simian virus 40).

The promoters in the commercially available yeast expression vectors may be active constitutively or inducible ones.

A constitutive promoter is a kind of promoter which is unregulated and allows continual transcription of its associated gene.

Example of a constitutive promoter: GAP promoter of the gene encoding glyceraldehyde-3-phosphate dehydrogenase.

An inducible promoter is the one which works in a regulated manner and the expression of genes associated with them can be switched on or off at a particular stage of development or at a certain point of time.

Examples of inducible promoters: AOX1, GAL1, GAL10, nmt1, nmt42, and nmt81.

The AOX1 promoter of the gene encoding alcohol oxidase. It is induced by methanol and is best-suited for expression of the protein in Pichia pastoris.

The GAL1 and GAL10 promoters are other examples. They are induced by galactose and are suitable for protein expression in Saccharomyces cerevisiae.

The nmt1, nmt42, and nmt81 promoters which are induced by thiamine for protein expression in Schizosaccharomyces pombe.

Reporter Gene

  • The reporter gene is responsible for the production of the protein which can be detected and quantified with the help of a simple assay.
  • They serve as a tool to measure the efficiency of the gene expression and also to detect the intracellular localization of the protein.
  • The rate of expression of the structural gene is dependent upon the regulatory sequences which are located upstream to it.
  • The rate of expression of the gene can be measured by replacement of its protein-encoding portion. Also, it can be fused to another gene which expresses another protein. The presence of this another protein can be easily identified.
  • Reporter genes are useful in the identification of promoters, enhancers, and other proteins or regulatory elements which bind to them.

The most commonly utilized reporter genes are:

  • It acts as a reporter in the presence of X- gal.
  • Its levels are easily detected by the intensity of colour which is produced. The intensity of the blue colour produced is quantified.

2. CAT (chloramphenicol acetyltransferase) encoding gene of E. coli

  • The CAT gene encodes chloramphenicol acetyltransferase.
  • The transferase enzyme is responsible for the transfer of acetyl groups from acetyl CoA to the recipient antibiotic, chloramphenicol

3. Luciferase encoding gene of firefly, Photinus pyralis

  • Luciferase is accountable for the oxidation of luciferin.
  • The oxidation of luciferin results in the emission of yellow-green light. The emission of light is easily detected irrespective of the low levels.

4. Green fluorescent protein (GFP) encoding gene of jellyfish, Aequorea victoria

  • GFP was discovered by Shimomura.
  • It is an autofluorescent protein with 238 amino acid residues produced by the bioluminescent jellyfish Aequorea victoria.
  • In GFP, β-barrel is formed by eleven β strands. An α- helix runs through the center. The chromophore is located in the middle of the barrel. The amino acid residues from 65 to 67 with sequence Ser-Tyr-Gly form the chromophore, p- hydroxybenzylideneimidazolinone, which is fluorescent. The chromophore fluoresces at a peak wavelength of 508 nm (green light) when it is irradiated with UV or blue light (400 nm).
  • GFP serves as a tool for determining protein localization.
  • It serves as a tag whereby it is fused with a protein whose expression is to be monitored. Basically, the subcellular localization of the protein is investigated.
  • Genetic engineering techniques help in the production of vectors which contain the coding sequence of the unidentified protein, X, cloned in the coding sequence of the GFP.
  • This fusion product of GFP-X can now be transfected into target cells and the expression, as well as the subcellular location of the X protein, can easily be monitored and detected.

Ribosome Binding Site and Translation Initiation Site

  • The ribosomal binding site (RBS) follows the promoter. It is responsible for the efficient translation of the cloned gene.
  • The translation initiation site in case of prokaryotes is known as the Shine Dalgarno sequence. This sequence is enclosed within the RBS only.
  • The consensus sequence of the translation initiation site includes a set of 8 base pairs present upstream the AUG start codon.
  • The translation in eukaryotes is initiated at a particular sequence called Kozak sequence.
  • The ribosomal machinery for the translation of mRNA is assembled on this site.

Polylinkers

  • Each vector contains particular recognition sites for restriction enzymes. It is at the restriction site that the vector is excised to clone the foreign gene of interest.
  • These sites often lie close together and, hence, are called polylinkers or multiple cloning sites (MCS).
  • These regions are 50 to 100 base pair in length and may have a cluster of up to 25 restriction sites.

Poly-A (polyadenylation) Tail

  • The poly-A tail present, at the end of the mRNA formed, protects the mRNA from degradation by the exonucleases or endonucleases.
  • It is extremely critical for the stability of the mRNA.
  • It is also responsible for the termination of transcription and translation and stabilizes the mRNA production.
  • A nucleolytic enzyme complex and a poly-A-polymerase are prerequisites for the addition of poly-A tail at the end of the mRNA.

Expression System

The production of a protein requires an expression system. There are two types of expression systems, prokaryotic and eukaryotic expression system. Each of them has its own advantages and drawbacks which can be taken into consideration while constructing an expression system. However, there is no such expression system which can be considered universal for the heterologous protein production.

Prokaryotic Expression System

  • The specificity of the promoter of an RNA polymerase, in the case of prokaryotes, is mediated by sigma factor.
  • E. coli is the widely used prokaryotic expression system.
  • It expresses high levels of the protein.
  • The E. coli strains are manipulated genetically for the production of recombinant protein so that they are rendered safe for large-scale experiments and fermentation.
  • The purification of the protein has become easier since recombinant-fusion proteins can be purified by affinity chromatography, say for example glutathione-S-transferase and maltose-binding fusion proteins.

Regardless of the advancements and improvements occurring,in the prokaryotic expression system, there are still many difficulties associated and challenges posed by the production of protein from the cloned foreign genes. These kinds of challenges can be grouped together into 2 categories:


Resilience

Reducing the effects of significant adversity on children’s healthy development is essential to the progress and prosperity of any society. Science tells us that some children develop resilience, or the ability to overcome serious hardship, while others do not. Understanding why some children do well despite adverse early experiences is crucial, because it can inform more effective policies and programs that help more children reach their full potential.

One way to understand the development of resilience is to visualize a balance scale or seesaw. Protective experiences and coping skills on one side counterbalance significant adversity on the other. Resilience is evident when a child’s health and development tips toward positive outcomes — even when a heavy load of factors is stacked on the negative outcome side.

Over time, the cumulative impact of positive life experiences and coping skills can shift the fulcrum’s position, making it easier to achieve positive outcomes. Play Tipping the Scales: The Resilience Game to learn more.

The single most common factor for children who develop resilience is at least one stable and committed relationship with a supportive parent, caregiver, or other adult. These relationships provide the personalized responsiveness, scaffolding, and protection that buffer children from developmental disruption. They also build key capacities—such as the ability to plan, monitor, and regulate behavior—that enable children to respond adaptively to adversity and thrive. This combination of supportive relationships, adaptive skill-building, and positive experiences is the foundation of resilience.

Children who do well in the face of serious hardship typically have a biological resistance to adversity and strong relationships with the important adults in their family and community. Resilience is the result of a combination of protective factors. Neither individual characteristics nor social environments alone are likely to ensure positive outcomes for children who experience prolonged periods of toxic stress. It is the interaction between biology and environment that builds a child’s ability to cope with adversity and overcome threats to healthy development.

Research has identified a common set of factors that predispose children to positive outcomes in the face of significant adversity. Individuals who demonstrate resilience in response to one form of adversity may not necessarily do so in response to another. Yet when these positive influences are operating effectively, they “stack the scale” with positive weight and optimize resilience across multiple contexts. These counterbalancing factors include

  1. facilitating supportive adult-child relationships
  2. building a sense of self-efficacy and perceived control
  3. providing opportunities to strengthen adaptive skills and self-regulatory capacities and
  4. mobilizing sources of faith, hope, and cultural traditions.

Learning to cope with manageable threats is critical for the development of resilience. Not all stress is harmful. There are numerous opportunities in every child’s life to experience manageable stress—and with the help of supportive adults, this “positive stress” can be growth-promoting. Over time, we become better able to cope with life’s obstacles and hardships, both physically and mentally.

The capabilities that underlie resilience can be strengthened at any age. The brain and other biological systems are most adaptable early in life. Yet while their development lays the foundation for a wide range of resilient behaviors, it is never too late to build resilience. Age-appropriate, health-promoting activities can significantly improve the odds that an individual will recover from stress-inducing experiences. For example, regular physical exercise, stress-reduction practices, and programs that actively build executive function and self-regulation skills can improve the abilities of children and adults to cope with, adapt to, and even prevent adversity in their lives. Adults who strengthen these skills in themselves can better model healthy behaviors for their children, thereby improving the resilience of the next generation.


Author information

Present address: Federal Environment Agency, Section IV 2.2 Pharmaceuticals, Washing and Cleansing Agents, and Nanotechnology, Wörlitzer Platz 1, 06844, Dessau, Germany

Affiliations

Department of Biochemistry, University of Bayreuth, Universitätsstr. 30, 95447, Bayreuth, Germany

Molecular Biology of Archaea, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Strasse, 35043, Marburg, Germany

Alexander Wlodkowski & Sonja-Verena Albers

Institute of Chemistry and Bioanalytics, School of Life Sciences, University of Applied Sciences of Northwestern Switzerland (FHNW), Gründenstrasse 40, 4132, Muttenz, Switzerland