Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics
© Brough et al; licensee BioMed Central Ltd. 2007
Received: 29 November 2006
Accepted: 10 May 2007
Published: 10 May 2007
The ability to regulate transgene expression has many applications, mostly concerning the analysis of gene function. Desirable induction characteristics, such as low un-induced expression, high induced expression and limited cellular heterogeneity, can be seriously impaired by chromosomal position effects at the site of transgene integration. Many clones may therefore need to be screened before one with optimal induction characteristics is identified. Furthermore, such screens must be repeated for each new transgene investigated, and comparisons between clones with different transgenes is complicated by their different integration sites.
To circumvent these problems we have developed a "screen and insert" strategy in which clones carrying a transgene for a fluorescent reporter are first screened for those with optimal induction characteristics. Site-specific recombination (SSR) is then be used repeatedly to insert any new transgene at the reporter transgene locus of such clones so that optimal induction characteristics are conferred upon it. Here we have tested in a human fibrosarcoma cell line (HT1080) two of many possible implementations of this approach. Clones (e.g. Rht14-10) in which a GFP reporter gene is very stringently regulated by the tetracycline (tet) transactivator (tTA) protein were first identified flow-cytometrically. Transgenes encoding luciferase, I-Sce I endonuclease or Rad52 were then inserted by SSR at a LoxP site adjacent to the GFP gene resulting stringent tet-regulated transgene expression. In clone Rht14-10, increases in expression from essentially background levels (+tet) to more than 104-fold above background (-tet) were reproducibly detected after Cre-mediated insertion of either the luciferase or the I-Sce I transgenes.
Although previous methods have made use of SSR to integrate transgenes at defined sites, none has effectively combined this with a pre-selection step to identify integration sites that support optimal regulatory characteristics. Rht14-10 and similar HT1080-derived clones can now be used in conjunction with a convenient delivery vector (pIN2-neoMCS), in a simple 3-step protocol leading to stringent and reproducible transgene regulation. This approach will be particularly useful for transgenes whose products are very active at low concentrations and/or for comparisons of multiple related transgenes.
The introduction of gene expression vectors into vertebrate cells is a powerful way to investigate gene function. Following transgene integration into the genome of a recipient cell, stable, heritable phenotypes are studied in cultured cells, or (if host cells are totipotent) in a developing/adult animal. Transgenes are typically used either to express or down-regulate a specific target gene, the latter being achieved via the use of RNA interference (RNAi) or site-specific recombinases (e.g. Cre) whose recognition sequences (e.g. loxP) have been engineered into the target gene. In cases where the gain or loss of gene function places host cells at a selective disadvantage, is lethal, or has different consequences at different developmental stages, temporally and/or spatially regulated transgene expression is necessary [1–4].
Temporally regulated transgene expression can be achieved with a variety of regulatory systems, but the most widely used are based on the tetracycline (tet) repressor TetR. The original Tet-Off system  is based on the tet-transactivator (tTA), a fusion protein between TetR and a minimal transactivation domain of the viral VP16 protein. In the absence of tet, tTA binds to tetO recognition sequences in the Tet Response Element (TRE) upstream of the gene to be regulated, and activates RNA polymerase II-dependent transcription from an adjacent minimal promoter (CMVmin). When tet is present, tTA dissociates from the TRE and transcription ceases. Many variations on the Tet-Off system have been developed (e.g. see [6–8]) often with the aim of improving stringency. A stringent system here refers to one with minimal transgene expression before induction and robust expression after induction.
Use of tet-regulated transgenes either to express or silence a target gene is already widespread in both cell lines and animals, but new applications continue to be developed. For example, with the growing use of site-specific recombinases [9, 10], RNA polymerase II-based methods for stably expressing short hairpin RNA (shRNA [11, 12]), and the expression of inducible transgenes in host cells with disrupted target alleles [13, 14], the scope for tet-regulated gene silencing is still expanding. While good stringencies are often described in such studies, these can be time-consuming to generate and, depending on the purpose of the study, may be less than ideal.
Chromosomal position effects  at the site of transgene integration are key variables in the success of these approaches as they can adversely affect the stringency of tet-regulation. As a direct result, position effects increase the effort required to identify cell or animal lines with sufficiently stringent transgene regulation, and complicate the comparisons between different lines. When evaluating lines expressing two related inducible transgenes, for example, any phenotypic variations can reflect differences either between the protein products of the transgenes or, more trivially, between their induced expression levels, as determined by the transgene integration sites. Dissimilar phenotypes can also indicate dysregulation of different host genes close to the transgene integration sites .
To date, the influence of position effects on tet-regulation has been limited in two different ways. One method uses insulator sequences  to protect the transgene from surrounding sequences (and vice versa). While this has been useful in increasing stringency , variation between different clones remains. In another approach, site-specific recombination (SSR) is used to integrate tet-regulated transgenes at specific chromosomal loci [19–22]. This approach effectively eliminates variations between clones, allowing for reproducible induction characteristics, but its success in improving stringency depends on the chosen integration site and this has not been systematically optimised.
Here we describe a new approach in which clones with optimal integration sites are first identified by flow cytometric screens of cells that have been stably transfected with a loxP-tagged reporter transgene (encoding enhanced green fluorescent protein [EGFP]). Cre-mediated recombination is then used to insert a promoterless gene of interest (GOI) at the loxP site of such clones. We have validated this approach, which we term "Screen and Insert' (or ScIn), using luciferase as a test GOI and two different implementation strategies. We have also used the method to generate clones that stringently regulate the expression of I-Sce I, an endonuclease widely used to study cellular responses to the formation of DNA double strand breaks (DSBs) [23, 24], and Rad52, a protein involved in the repair of DSBs by homologous recombination .
Screen and insert: principles and implementation strategies
In the present study we have tested two of many possible ways in which the basic ScIn strategy can be implemented. We will refer to these as ScIn-1 and ScIn-2 and their key features are summarised in Fig. 1B and 1C, respectively. The target construct (pTARG4) used for both ScIn-1 and ScIn-2 carries a destabilised EGFP reporter gene (d2EGFP) driven by an optimised TRP (from pTRE-tight, Clontech). Positioned between the TRP and d2EGFP are a recognition site (FRT) for the Flp site-specific recombinase, and a mutant loxP site, lox71. The FRT site is necessary for ScIn-2 only. Lox71 was chosen because it is known to undergo unidirectional Cre-dependent recombination with lox66 . Thus, by use of a lox66 site in the insertion construct, unimolecular Cre-mediated excision events that reverse the desired integration events, are minimised.
In ScIn-1, the GOI (e.g. luciferase) is linked to a promoterless drug resistance marker gene (e.g. hyg) by an internal ribosome entry site (IRES). With this arrangement it is possible to select in drug (hygromycin) for the desired insertion events. A limitation of ScIn-1 is that for drug selection to work the GOI must also be expressed. In ScIn-2 this limitation is avoided by use of an insertion construct in which a promoterless drug-resistance gene (e.g. gpt) is linked to the GOI (luciferase) by an FRT site. This arrangement allows insertion events to be selected in drug (Mycophenolic acid and xanthine [MPA/X]) without any accompanying GOI expression. Efficient Flp-mediated excision is then used to delete the drug-resistance marker and bring the GOI under the control of the TRP, in the presence of tet.
Screening: identification of clones with stringently regulated target constructs
An initial target construct (pTARG1) used the original TRE . Of over 100 puror pTARG1 transfectants, many showed good regulation, but even the best expressed significant uninduced levels of GFP and required 144 h for full down-regulation (e.g. clone 6, Fig. 2B). To facilitate screening we used a second target construct (pTARG2) with d2EGFP as reporter. This reporter has reduced half-life compared to EGFP. Over 200 puror pTARG2 transfectants were screened, but none completely silenced GFP expression in the presence of tet. Flow cytometric profiles of the best-regulated clones are shown in Fig. 2C. When a target construct (pTARG3) with an improved TRE was used, 8 of 63 puror clones analysed had uninduced GFP expression levels of less than twice the background level, but these showed heterogeneous GFP expression in the absence of tet (Fig. 2D). Experiments with the methylation inhibitor 5-aza-2'deoxycytidine (AZC) indicated that heterogeneous expression was caused by DNA methylation of the tTA gene . For example, when T15, one of the clones transfected with pTARG3, was treated with AZC for 24 h, a marked increase in GFP-expressing cells was observed (Fig. 2E). Furthermore, luciferase expression in the same clone transiently transfected with a luciferase reporter gene linked to the improved TRE (pTIGHT-luc, Clontech) was also stimulated (2.6-fold) by a 24 h treatment of the cells with AZC prior to the transfection (Methods). This latter experiment suggested that methylation of the tTA gene, not the TRE, was responsible heterogeneous expression. The relatively homogeneous GFP expression seen in pTARG1 and pTARG2-transfected clones (Fig. 2C,D) probably reflects the use of lower passages of HT2 cells that were less likely to have suffered tTA gene silencing.
To minimise tTA gene methylation, the itTA-expressing HT1080 line Rht14 was made (Methods) and used for a final screening experiment. To allow subsequent testing of both ScIn-1 and ScIn-2, an FRT-tagged target construct (pTARG4) was used. Of 32 puror clones analysed, 7 had uninduced GFP expression levels of <2-fold above background combined with acceptable single-peak profiles (Fig. 2F), while others showed heterogeneous expression (Fig. 2G). The two best clones (Rht14-10 and Rht14-19) were chosen for further analysis.
Both Rht14-19 and Rht14-10 were found by Southern blot analysis (not shown) to carry a single copy of pTARG4. To test for stability of expression, clone Rht14-10 was cultured continuously for 42 days during which time the profile of GFP expression remained largely unchanged (Fig. 2H). A comparison of flow cytometric and immunoblot analyses of tet-induced GFP down-regulation in clone Rht14-19 illustrates one of the advantages of screening by flow cytometry, namely its sensitivity (Fig. 2I). Thus, substantial above-background GFP expression, readily detectable by flow cytometry, is undetectable on immunoblots. A second advantage of flow cytometric screening is its ability to detect and eleminate heterogeneously expressing clones, such as those in Fig. 2D and 2G, which would have been difficult or impossible to identify by bulk measurements of GFP expression (e.g. by immunoblot).
Frequencies of Cre-mediated insertion events
a Host cell
For Flp-treated 19IN5 cells, 12 pools (6 clones/pool) were assayed by PCR (Fig. 5C). Two pools (9 and 12) were positive, each due to the presence of a single clone 19flp9.2 and 19flp12.3, respectively. The relatively low efficiency of Flp-mediated deletion in 19IN5 (0.03 [2/72] events per transfected cell) probably reflects a lower efficiency of pCAGGS-flpe delivery due to the inclusion of tet in lipofection mix of that particular transfection. Southern blot (Fig. 4C) and PCR analysis (Fig. 5C), showed that 19flp9.2 was impure, the majority of cells being of parental (19IN5) type. Southern analysis of the other flp-deleted clone (19flp12.3), however, confirmed that it had undergone the expected deletion (Fig. 4C). Clone 19flp12.3 showed highly stringent tet-regulation of luciferase expression with an induction ratio of more than 10,000-fold and uninduced expression levels barely above background (Fig. 5D).
Use of ScIn-2 to generate cells with inducible I-SceI or Rad52 expression
Two clones, 10IN-SCE.1, 10IN-R52.1 (with pIN2-neoSCE and pIN2-neoRAD52 integrated, respectively) were grown in +tet medium, lipofected (- tet for 4 h; see methods) with pCAGGS-flpe and plated at low dilution in +tet medium. The majority of the resulting colonies were positive in a PCR assay for the deletion event, suggesting highly efficient Flp-mediate deletion (Fig. 6C–E). Two clones of high purity (10IN-SCE.1flp1, 10IN-R52.1flp1) as judged by PCR were analysed further. Immunoblot analyses confirmed the expected tet-regulated expression of the appropriate GOI (I-Sce I or Rad52) in these clones (Fig. 6F). Expression in the presence of tet was undetectable indicating, as far as is possible by immunoblotting, highly stringent tet-regulation.
Frequencies of G418r colony formation in pDRneo-transfected cells
Frequency of G418r
a 10IN-SCE.1flp1/DRneo, Clone 1
4.7 × 10-6
7.2 × 10-2
1.5 × 104
a 10IN-SCE.1flp1/DRneo, Clone 2
1.3 × 10-6
2.9 × 10-2
2.2 × 104
a 10IN-SCE.1flp1/DRneo, Clone 3
1.0 × 10-6
3.5 × 10-2
3.5 × 104
1.3 × 10-5
1.4 × 10-5
The "Screen and Insert" (ScIn) methods described here provide convenient and reproducible ways to isolate clones in which transgene expression is stringently regulated by tet. A great variety of modifications and improvements to the original Tet-Off system have been reported. Versatility, convenience, stringency and stability have been variously extended by the development of the reverse tTA (Tet-On) system , the combined use of tet-regulated activation and repression [34, 35], systems for single-vector delivery and/or autoregulation [36–44] and by modifications to the TRE [45–48] or the transactivator protein/gene [18, 28, 49–52]. Despite such improvements, the tet system remains subject to the influence of chromosome position effects, complicating the isolation of optimally regulated clones. The ScIn methods address this problem.
The key to ScIn is the combination of a powerful GFP-based screen to identify optimal chromosomal loci, and the use of SSR to target the insertion of transgenes at such sites. Screening by flow cytometry is valuable because it allows lines with even very small amounts of leaky expression, or with cellular heterogeneity of expression, to be eliminated. Furthermore, for a given cell line, one can envisage collecting a range of clones with different induction characteristics, from which one can choose the clone most suitable for a particular application. The ease with which many clones can be screened increases the chances of finding one in which optimal regulation is achieved. Although we screened clones individually here, it may be even more convenient in future to use flow sorting to purify and clone the best cells from a population of transfectants.
It is important to note that, once screening has been used to identify a clone (e.g. Rht14-10) with a stringently regulated reporter, the use of SSR to insert the GOI at the reporter locus is relatively labour-unintensive and can be used repeatedly for different GOIs. In ScIn2, for example, the following three steps are now sufficient to generate an HT1080 clone with any GOI under highly stringent tet-regulation: i) Clone GOI into multiple cloning site of pIN2-neoMCS and co-transfect with pMC-Cre into Rht14-10, selecting for G418r colonies. ii) Expand a G418r/GFP- colony, confirm G418r is tet-sensitive (optional), transfect with pCAAGflpe (or similar) and plate at low density in tet. iii) Identify one of the >10% of colonies that is no longer G418r when tet is removed.
ScIn has at least three advantages over the standard approach of immunoblot-screening of multiple clones with randomly integrated GOI. First, no screening for GOI expression is required to identify the desired clone. Second, tet-regulation of the GOI is likely to be much more stringent and lacking in cellular heterogeneity. Third, the same protocol can be used repeatedly with different GOIs to generate clones whose phenotypes can be compared without the complication of differential expression levels. These advantages will be attractive for a range of studies, including in both gain- and loss-of-function experiments. The expression of I-Sce I provides a good illustration of the former. Leaky expression of this endonuclease will lead to repeated cutting and repair of any I-Sce I recognition sites in the host genome, until inaccurate repair by non-homologous end-joining  results in the loss of I-Sce I sites. The ability to induce DSB, and measure cellular responses, will therefore be irreversibly lost if expression is leaky. The importance of minimal leakiness in loss-of-function experiments is illustrated by systems in which a tet-regulated transgene is expressed in cells whose corresponding endogenous alleles have been inactivated [13, 14]. Switching off the tet-regulated transgene in such systems will produce a true null phenotype only when there is no leakiness.
Success of the ScIn method depends on optimal reporter gene regulation from the target construct and this was achieved best with pTARG4. Stringent regulation from the other contructs (pTARG1-3) was limited by use of the original TRE and/or by host cells that expressed tTA rather itTA. Although we saw no signs of structural instability or unstable gene expression at pTARG4 loci, the persistence of prokaryotic vector sequences at transgene loci can in some circumstances be problematic [54–56]. Removal of unwanted vector DNA from target and insertion constructs prior to transfection might therefore be considered desirable. For target constructs this may be achieved simply by digestion with appropriate restriction enzymes. For insertion constructs, which must remain circular, this might be achievable by inclusion of a second loxP site and use of Cre recombinase in vitro.
The use of SSR to integrate transgenes at sites that support tissue-specific or ubiquitous expression in mice is established [57, 58]. For tet-regulated expression, SSR has been used to target either to defined loci, such as the HPRT  or ColA1  genes in mouse embryonic stem (ES) cells, and the DHFR locus in Chinese Hamster Ovary (CHO) cells locus, or to specific but undefined loci in e.g. CHO , human cervical  and rat pancreatic cells . While these systems introduce the valuable aspect of reproducibility into transgene integration in a given cell line, the chosen target sites are not necessarily optimal for stringency or for cellular homogeneity of induction. Furthermore, establishing these systems in new cell lines is demanding.
In an approach more closely related to ScIn, Puttini et al  used I-Sce I-stimulated homologous recombination (HR) to target transgenes to a locus that was identified by its ability to support stringent tet-regulation of another reporter, secreted alkaline phosphatase (SEAP). In practice, this approach was limited by the low efficiency of HR-mediated integration, the inability to use SEAP assays to assess cellular heterogeneity and the presence of a promoter/enhancer close to the TRE. In another approach related to ScIn, a tet-regulated gpt gene was linked to a promoterless GOI and randomly integrated into the genome . After selecting clones with optimally-regulated gpt, SSR was use to invert the DNA segment carrying gpt and GOI, placing the GOI under control of a TRE. This system suffered from the low stringency of gpt selection and the need to select optimally-regulated clones for each new GOI.
Emphasis in this paper has been on the use of transgenes for the analysis of gene function in cultured cells. The ScIn may also be valuable when extended to mice. For this, however, methods must be developed to screen for ES cell clones that support tet-regulated GFP expression not just in culture, but also in all tissues of ES-derived animals. Provided its tTA/rtTA gene can be deleted (e.g. by SSR), the chosen clone can then be used repeatedly for the targeted insertion of any GOI, and the generation of mice to be crossed with transgenic lines chosen for their tissue-specific expression of tTA/rtTA.
The screen and insert approach described is a highly effective way to achieve stringent and reproducible tet-regulated transgene expression. Reagents developed here (e.g. clone Rht14-10 and the insertion vector pIN2-neoMCS) can be used immediately in a simple 3-step protocol to achieve such expression in an HT1080 background. Furthermore, additional reagents we describe (e.g. pTARG4), can be used to establish the select and insert method in other cell lines. The method is particularly attractive for analyses of gene function in situations where the gene product is likely to have biological effects even when expressed at very low levels and/or where the effects of several related genes are to be compared. Cell lines expressing stringently regulated I-Sce I or Rad52, generated in the course of this study, will be useful in studies of DNA damage and repair.
A lox71 site (annealed oligonucleotides 5'-TACCGTTCGTATAGCATACATTATACGAAGTTAT-3' and 5'-CTAGATAACTTCGTATAATGTATCGTATACGAACGGTAGC-3') was ligated to the Sac II and Xba I sites of pUHD10-3 (, kindly donated by H. Bujard, ZMBH, Heidelberg) to create pTRElox71. To generate pTARG1, an Xba I fragment from pEGFP (Clontech), carrying the EGFP open reading frame (ORF), was inserted at Xba I site downstream of the lox71 site of pTRElox71.
The ORF of d2EGFP (from pd2EGFP, Clontech) was isolated as a Not I/Age I fragment and inserted into the Not I/Age I sites of pTRElox71.
A lox71 site (annealed oligonucleotides 5'-AATTCTACCGTTCGTATAGCATACATTATACGAAGTTATACTAGTG-3' and 5'-GATCCATCAGTATAACTTCGTATAATGTATGCTATACGAACGGTAG3') was cloned into the EcoR I and Bam HI sites of pTRE-TIGHT (Clontech), creating pTlox71. To generate pTARG3, the ORF of d2EGFP (from pd2EGFP, Clontech) was isolated as a Not I/Bam HI fragment and inserted into the Not I and Bam HI sites of pTlox71.
An FRT site (annealed oligonucleotides 5'-AATTAGATCTGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC-3' and 5'-AATTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCAGATCT-3') was cloned into the Hind III/Nco I sites of pTARG3.
A lox66 site (annealed oligonucleotides 5'-TCGAGAATTCATAACTTCGTATAGCATACATTATACGAACGGTAG-3' and 5'-AGCTCTACCGTTCGTATAATGTATCGTATACGAAGTTATGAATTC-3') was cloned into the Xho I/Hind III sites of pGL3-Basic (Clontech) to generate plox66luc. To generate pIN-1, pIRESHyg3 (Clontech) was digested with Xho I/Nhe I and the IRES-Hyg fragment was cloned into the Xba I/Sal I site of plox66luc, downstream of the luciferase ORF.
The gpt ORF was removed from pBSgpt  as a Bam HI/Bgl II fragment and cloned into the Bgl II site of pGL3-Basic (Clontech) to make pGL3gptluc. A lox66 site (annealed oligonucleotides 5'-CTAGATAACTTCGTATAGCATACATTATACGAACGGTAGAAT-3' and 5'-TCGAATTCTACCGTTCGTATAATGTATGCTATACGAAGTTAT-3') was then cloned into the Nhe I/Xho I sites of pGL3gptluc upstream of the luciferase ORF to make plox66gptluc. To generate pIN-2, an FRT site (annealed oligonucleotides 5'-AGCTGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGAATT-3' and 5'-CATGAATTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTC-3') was cloned into the Hind III/Nco I sites of plox66gptluc located between the luciferase and gpt ORFs.
An MCS (annealed oligonucleotides 5'-CGCTAGCAGCTGGTCCGCGGACTAGTCCCGGGAAGCTTCTCGAGAGGCCTCATATGCATGCCATGGCCGG-3' and 5'-CCATGGCATGCATATGAGGCCTCTCGAGAAGCTTCCCGGGACTAGTCCGCGGACCAGCTGCTAG-3') was inserted into Bst BI/Fse I sites of pIN-2, removing the luciferase ORF, to create pIN2-MCS. pSV2neo (Southern et al., 1982) and plox66gptLuc were cut with Bgl II and Bam HI and neomycin ORF was cloned downstream of the lox 66 site to create plox66Neo. To create pIN2-neoMCS, pIN2-MCS and plox66neo were cut with Bgl II and Pfm I and the gpt ORF was replaced with the neomycin ORF.
pIN2-neoSCE and pIN2-neoR52
pIN2-neoMCS was cut with Eco RI, end-filled then cut with Sal I. pFB580 (human RAD52 cDNA cloned into pUC18, a kind gift from F. Benson and S. West, Cancer Research UK, London) and pCMV3xnls-I-Sce I (, a kind gift from M. Jasin, Sloane Kettering Institute, New York) were also cut with Eco RI, end-filled then cut with Sal I, to release the RAD52 and I-Sce I ORFs, respectively. RAD52 and I-SceI ORFs were then cloned into pIN2-neoMCS to generate pIN2-neoR52 and pIN2-neoSCE, respectively.
plox66Neo (see above) and pSV2neo were digested with Bgl II and Bam H1 and the neo ORF was cloned downstream of the lox66 site.
A plasmid (pRK5-itTA) encoding the improved transactivator itTA  was kindly donated by R. Sprengel, Max-Planck-Institute, Heidelberg. The itTA ORF was cloned as an Eco R1/Bcl I fragment into the Eco RI/Bcl I sites of pZeoSV (Invitrogen), to create pZeoSVitTA. pTIGHTgpt was made by inserting the gpt ORF as a Bgl II/Bam H1 fragment from pBSgpt into the Bam H1 site of pTRE-TIGHT.
Conditions used for the culture of HT1080 cells and derivatives have been described previously . When required the medium was supplemented with one or more of the following drugs: 5-azacytidine (1 μM), hygromycin B (100 μg/ml), mycophenolic acid (10 μg/ml), puromycin (0.4 μg/ml), tetracycline (1 μg/ml), xanthine (100 μg/ml) and zeocin (200 μg/ml). HT-2 cells have been described  and were also called HTET . A telomerase-immortalised human retinal epithelial cell line (hTERT-RPE1; Clontech) was cultured in a similar manner to HT1080 cells.
Cell lines generated by electroporation
Electroporation with a Gene Pulser (BioRad) was as described . Rht14 cells were made by electroporation of HPRT+ HT1080 cells with 10 μg Bgl II-linearised pZeoSVitTA. Zeocinr colonies were screened by lipofection (see below) with pTIGHTLuc (Clontech) grown in the presence or absence of tetracycline. After 48 hours, lysates were prepared and assayed for luciferase. Rht14 was chosen from among 24 colonies for its low luciferase activity in the presence of tet and its high induction ratio. Co-electroporation was used to generate clones with randomly integrated target plasmids. Target plasmid (20 μg) was linearised with Pvu I (pTARG1) or Bgl I and Ssp I (pTARG2, pTARG3, pTARG4), gel-extracted, ethanol-precipitated and co-electroporated with 1 μg of similarly purified, Spe I-linearised pBL-PuroR , into Rht14 cells. Puror colonies identified as GFP-positive by fluorescence microscopy (Zeiss Axiovert S100TV microscope) were picked for further analysis.
Lipofectamine 2000 (Invitrogen) was used for delivery of insertion constructs and transient expression of Cre and Flp recombinases and luciferase. 24 hours prior to transfection, 250,000 cells were plated into 6-well plates in 2 ml of antibiotic-free (- tet, unless otherwise stated) medium. On the day of transfection a DNA/Lipofectamine/OptiMEM mix was prepared (0.5 ml/well) according to the manufacturer's instructions and incubated with the cells for 4 hours, after which the mix was replaced with fresh medium. For Cre-mediated insertion, 2 μg of insertion construct and 2 μg of pMC-Cre15 (kindly donated by H. Gu, University of Köln) were used per well. The following day, the cells were seeded into 9 cm plates (105 cells per plate) and the appropriate drug selection was added after a further 24 hours. For flp-mediated excision, 4 μg of pCAGGS-flpe (Cambion) was used per well and cells were plated the following day at low density (50–500 cells per 9 cm [diam] dish) in medium with tet. To analyse the effect of AZC on luciferase expression from pTIGHT-luc, clone T15 cells were grown with or without AZC for 24 h, then plated in antibiotic-free medium and lipofected 24 h later (as above) with pTIGHT-luc (4 μg). After a further 24 h lysates were prepared and assayed for luciferase (see below).
Cells were grown in 6-well plates with or without tet for 48–72 hours (except where longer times are indicated) prior to FACS analysis. Cells, generally no more than 80% confluent on the day of analysis, were trypsinised and resuspended in cold phosphate buffered saline (PBS) at a density of 1000 cells per μl. 40,000 cells were analysed in a FACScan machine (Becton Dickinson) with an argon laser tuned to 488 nm (FL-1; with fluorescence channel FL-3 as a control). Acquisition, storage, and analysis of data were carried out using CellQuest software (Becton Dickinson).
Immunoblots were carried out as described previously for Rad52 . For GFP detection, a primary monoclonal antibody was used (Clontech, 632380; 1/1000 dilution) and a secondary, horseradish peroxidase-conjugated goat anti-mouse immunoglobulin antibody (Sigma, P-0447; 1/1000 dilution) was used. For HA-tagged I-Sce I detection, the primary antibody was a rat monoclonal (Roche, 3F10; 1/500 dilution) and the secondary a horseradish peroxidase-conjugated goat anti-rat immunoglobulin antibody (Sigma, A-9037; 1/1000 dilution)
Polymerase Chain Reactions (PCR)
Cell pellets (100–10,000 cells) were resuspended in a 25 μl Taq polymerase buffer (Qiagen) containing pronase (0.6 μg/μl), incubated at 50°C for one hour, 95°C for 10 minutes and then placed on ice. A mix (25 μl) containing nucleotides (Pharmacia, 0.5 μM), Taq polymerase (Qiagen, 1.25 U) and oligonucleotides (100 ng each) dissolved in in Taq polymerase buffer was added. Annealing temperatures (Ta) for the various primer pairs were 60°C (O2/O4 and O1/O3), 63°C (O2/O5), and 57°C (O2/O6 and O2/O7). Temperature sequences were: 95°C (10 min.) then 30 cycles of 95°C, Ta, and 72°C (1 min each) then 72°C (10 min.). Primers were: O1 (5'-ACGAGGCCCTTTCGTCTTCA-3'), O2 (5'-TTTAGTGAACCGTCAGATCGCC-3'), O3 (5'-CCACGGCTTACGGCAATAATGC-3'), O4 (5'-CCCCTTTTTGGAAACGAACAC-3'), O5, (5'-AGAAGGCGATAGAAGGCGATGC-3'), O6 (5'-ACTCGAACTGCATACAGTAG-3') and O7 (5'-CCAAAGATAAAGCCTTGGAC-3').
As positive controls for the DNA template, ~10 pg of the indicated plasmid DNA, or genomic DNA from 1000 pelleted cells of the indicated cell line, were used. TetNeo cells have a tet-regulated neo gene and are described in detail elsewhere . Briefly, plox66Neo was inserted by Cre-mediated recombination into tTERT-RPE1 cells with an integrated pTARG4 supporting tightly regulated GFP. PCR products were analysed by standard agarose gel electrophoresis with 1 kb ladder marker DNA (Invitrogen).
Standard methods were used, as previously described . The GFP probe was a 727 bp Nco I fragment from pd2EGFP [Clontech]). Marker DNA was end-labelled 1 kb ladder (Invitrogen).
A commercial kit (Promega) was used according to manufacturers instructions. Briefly, cells were seeded at 105 cells per 3.5 cm (diam.) well, incubated in medium with or without tet for 48 hours, then washed with PBS and agitated gently for at least 20 minutes with 500 μl passive lysis buffer. The cell lysate was cleared by centrifugation and 20 μl added to 100 μl of luciferase assay reagent in a luminometer tube. The relative light units (RLU) were measured immediately in a Biorbit 1253 luminometer. The average of ten RLU measurements taken at 2-second intervals was recorded.
Recombination of pDRneo
To estimate the recombination frequency when I-Sce I was not expressed, 3 million cells were distributed in three 15 cm (diam.) plates and selected in G418 and tetracycline and colonies counted after 14 d. To determine the frequency after I-Sce I induction, cells were grown in the absence of tet for 48 h and then plated (at 50, 100, 250 cells per 9 cm [diam.] plate) in G418 and tetracycline and colonies counted after 14 d. To account for the plating efficiency, the proportion of G418r clones was calculated as a fraction of the total number of colonies formed when compared to the amount generated on equivalent unselected (no G418) plates. Rht14/DRneo cells were analysed in the same way except that tet was either present or absent from the growth medium throughout, and for 48 h prior to, G418 selection.
We are grateful to Fiona Benson, Herman Bujard, Maria Jasin, Rolf Sprengel and Steve West, for generously supplying reagents. This work was funded by the MRC.
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