Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.
When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2–0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.
When generating transgenic plants, usually the objective is to have the integrated transgene(s) stably expressed. This can be achieved by intact single copy integrations of a transgene. Multiple integrations can lead to increased expression of a transgene, but also to gene silencing. Silencing of transgenes is more likely if inserted sequences are arranged in tandem or inverted repeats1,2,3,4. Binary vectors are used as shuttles in Agrobacterium-mediated transformation experiments to deliver the sequences of interest into plant genomes. The number of integrations into a plant genome is dependent on the copy number of the binary vector in Agrobacterium tumefaciens5,6. Many commonly used binary vectors are high copy vectors, and therefore yield a high average transgene copy number: 3.3 to 4.9 copies in Arabidopsis5.
The number of T-DNA integrations can be lowered by using binary vectors that have a low-copy number in A. tumefaciens, such as BIBAC7, or by launching a T-DNA from the A. tumefaciens chromosome5. The average number of transgene integrations in such cases is below 25,8,9,10. Due to being single-copy in A. tumefaciens, and also in Escherichia coli, BIBAC-derivatives can maintain and deliver constructs as large as 150 kb11.
GW-compatible BIBAC vectors10,12 allow easy introduction of genes of interest into the vector using Gateway cloning. The use of Gateway technology greatly simplifies the cloning procedure, but also overcomes common problems associated with large low-copy-number vectors13,14, such as a low DNA yield and a limited selection of unique restriction sites available for cloning7,11. The pBIBAC-GW derivatives are available with either resistance to Glufosinate-ammonium (pBIBAC-BAR-GW) or DsRed fluorescence in seed coats (pBIBAC-RFP-GW) for selection in plants (Figure 1)10,12. For both vectors, a kanamycin resistance gene is used as the selection marker in bacteria.
The pBIBAC-GW vectors combine: (1) easy design and genetic manipulation in E. coli, and (2) intact single-copy integrations in planta at high efficiency. The pBIBAC-GW vectors yield on average 1.7 integrations in Arabidopsis with approximately half of the transgenic plants carrying a single integrated T-DNA10.
Stable expression of transgenes is a requirement for most transgenics generated. Stable transgene expression can be achieved by intact, single-copy integrations. Working with transgenic plants carrying intact, single-copy integrations is, however, even more important if for example, the aim is to study the efficiency of chromatin-based processes, such as mutagenesis, recombination, or repair, and the dependence of these processes on the genomic location and the chromatin structure at the insertion site. For our interest, to study the dependence of oligonucleotide directed mutagenesis (ODM) on the local genomic context, a set of reporter lines with intact, single-copy integrations of a mutagenesis reporter gene was generated (Figure 2)10. Using this set of lines, it was shown that the ODM efficiency varies between transgenic loci integrated at different genomic locations, despite the transgene expression levels being rather similar.
1. Inserting Sequences of Interest into Binary Vector
2. Preparation of A. tumefaciens for Floral Dipping of Arabidopsis
3. Arabidopsis Transformation
4. Characterizing Transgenics for the Number and Integrity of T-DNA Integrations
Using the BIBAC-GW system, reporter constructs for studying ODM in plants were generated10. Constructs were designed in the Gateway Entry vector pENTR-gm12 and inserted into pBIBAC-BAR-GW (Figure 1) using the Gateway LR recombination reaction.
Arabidopsis were transformed with pDM19, a BIBAC-BAR-GW plasmid with an mTurquoise-eYFP reporter carrying a translational stop codon in the eYFP reading frame at position 120 (mTurquoise2-eYFP*40) (Figure 2)10. In total, 126 Arabidopsis plants were transformed (9 plants per pot, 14 pots). Seeds of these plants were pooled, sown on trays with soil, and allowed to grow for two weeks prior to treatment with Glufosinate-ammonium solution. Only seedlings expressing the bar gene (present in BIBAC-BAR-GW) survive Glufosinate-ammonium treatment (Figure 3). In total, 11 transgenics transformed with pDM19 were identified, corresponding to a transformation efficiency of 0.02% of the seeds analyzed.
For the 11 transgenics isolated, DNA blotting was used to determine the number of T-DNA integrations. For that purpose, genomic DNA was cut with either BglII or ScaI (strategy as devised on Figure 4A). Both of these restriction enzymes cut only once into the T-DNA sequence (Figure 7A). Hybridization with probes recognizing the bar and eYFP coding regions allowed detection of the number of respective DNA fragments.
The number of individual DNA fragments on the blots allowed for estimating the number of T-DNA insertions in the reporter lines (Table 1). Single hybridizing fragments with both the Bar and eYFP probe indicated the presence of a single T-DNA integration. From the 11 transgenics analyzed, six carried single integrations. The average number of integrations was 1.2.
For 6 lines carrying a single T-DNA integration, the integrity of the inserted reporter construct was tested using DNA blotting (strategy as devised on Figure 4B). Genomic DNA was cut with BglII and PciI to release a 5.5 kb fragment containing both the Bar and mTurquoise-eYFP fusion gene (Figure 7A). A probe against eYFP was used to detect the expected fragment. All plants tested carried an intact fragment. Note that the fragment examined excludes the Left and the Right T-DNA border, and therefore does not examine the integrity of the entire T-DNA, but only the part containing the transgenes of interest.
The expression of the fluorescent reporter gene was determined in independent single-copy transgenic lines differing only by the genomic location of the T-DNA. Relative transcript levels of the CaMV-35S promoter-driven mTurquoise-eYFP reporter were measured by RT-qPCR in four DM19 reporter lines carrying intact, single-copy integrations of which the genomic position was determined10. The variation in reporter gene expression levels between the lines was minor: the maximum difference in mTurquoise-eYFP RNA levels was 2-fold (Figure 8A).
Next, the ODM was carried out in these reporter lines. Three out of the four independent reporter lines showed rather similar ODM efficiencies (Figure 8B). However, one line, DM19[4]1, yielded a very low ODM efficiency compared to the other lines. These results indicate that the ODM is affected by the local genomic context. In what manner the local genomic context of the T-DNA integration in DM19[4]1 differs from that in the other lines remains to be identified. Analysis of available datasets on active and inactive chromatin marks at the genomic T-DNA integration sites in non-transgenic plants did not provide an answer10.
Figure 1: Functional maps of pBIBAC-GW vectors. pBIBAC-GW derivatives are available with either resistance to Glufosinate (bar) or DsRed fluorescence in seed coats (DsRed) as a selection marker in plants. For both vectors, a kanamycin resistance gene is the selection marker in bacteria. The Gateway ccdB cassette is shown between green arrowheads representing recombination sites attR1 and attR2. Please click here to view a larger version of this figure.
Figure 2: Mutagenesis reporter construct. The mTurquoise-eYFP reporter genes are driven by the 35S-CaMV promotor. The mTurquoise coding region is fused to an eYFP coding region carrying a C-A mutation at nucleotide position 120, resulting in a premature translational stop codon TAA, and premature termination of the translation of the fusion protein. The 3′ Nopaline Synthase (3'nos) polyadenylation signal is used to terminate the transcription of the construct22. Nuclear localization signal (NLS) is used to target the translated proteins to the nucleus. Please click here to view a larger version of this figure.
Figure 3: Tray filled with Arabidopsis seedlings before and after Glufosinate-ammonium treatment. Seedlings not expressing the bar gene that is present in the pBIBAC-BAR-GW T-DNA die after being sprayed with Glufosinate-ammonium solution. The photos show the same tray of seedlings (A) before spraying with Glufosinate-ammonium, 14 days after sowing, and (B) 10 days later, after being sprayed twice. Please click here to view a larger version of this figure.
Figure 4: General DNA restriction strategy to identify the number and intactness of inserted T-DNAs. (A) One restriction site (R) in the middle of the T-DNA allows independent probing of the left (red L) and right part of the T-DNA (green R). The cartoons on the right show that depending on single- or multi-copy T-DNA integrations, different banding patterns are obtained with DNA blotting. Bands marked with an * have a defined length, while the length of other bands depends on the closest restriction site in the flanking genomic DNA. Single insert: The L and R probe both give one independent fragment. The expected average fragment size can be calculated based on the frequency of the restriction site in the genome. The minimal size is the distance from the restriction site to the Left Border (LB) or Right Border (RB), depending on which end of the integration is being probed, and if the T-DNA is intact. Tandem repeat: The probes for L and R give both two fragments; for each probe one of the fragments includes flanking genomic DNA, the second fragment has an expected size and is identified by both probes. Inverted repeat: Depending on the directionality of the integrated cassette, either one L and two R fragments, or two L and one R can be identified. Individual single insertions: The result is a number of independent fragments, and the number of fragments corresponds to the number of integrations. (B) Restriction sites at the extremities of the T-DNA allow determining the integrity of the fragment between the restriction sites. Please click here to view a larger version of this figure.
Figure 5: An agarose gel with restriction pattern and the matching transparency. (A) On the agarose gel, genomic DNA digested with EcoRI is shown. The proper digestion of the DNA is illustrated by the presence of discrete satellite bands. (B) Marking the position of the slots and marker bands on a transparency makes it possible to later easily calculate the size of hybridizing fragments. Here, MRC Holland markers (Blue and Red) are used, indicated by M. Please click here to view a larger version of this figure.
Figure 6: Setup for capillary blotting. In a capillary blotting setup, filter paper is placed on a plastic plate with the ends of the paper hanging in 20x SSC buffer. The paper is wetted with 20x SSC, and an agarose gel placed on top, followed by a nylon membrane, filter paper, and a stack of tissues. A light weight is placed on top. Care is taken to remove air bubbles between the gel, paper and membrane. Cling film is used to avoid drying out of the setup. Please click here to view a larger version of this figure.
Figure 7: Example of DNA blotting strategy and experimental outcome. (A) DNA blotting strategy to determine the number and intactness of T-DNA integrations. Cutting locations of the selected restriction enzymes within the T-DNA are indicated with vertical bars. The eYFP and bar probes used for hybridization with digested genomic DNA are indicated using a line with the terminal dot below the T-DNA. (B–D) Example DNA blots. Genomic DNA was cut with ScaI and the blot was probed with both a bar and eYFP probe (B and C). Genomic DNA was cut with BglII and PciI and probed with a bar probe. Intact fragments are 5.5 kbp in size (D). Note that the set of samples in D differs from those shown in B and C. * indicates the expected fragment size; M, marker. In B, C, and D the same size marker is used. Please click here to view a larger version of this figure.
Figure 8: mTurquoise-eYFP expression levels and ODM efficiencies in independent mTurquoise-eYFP reporter lines. (A) Relative mTurquoise-eYFP transcript levels measured by RT-qPCR in DM19 reporter lines. For normalization transcript levels of Actin were used. (B) ODM efficiency measured in the DM19 reporter lines. For A and B, bars indicate the average of at least five biological replicates. Error bars indicate SEM. Please click here to view a larger version of this figure.
Type of T-DNA locus | Nr of T-DNA integrations | Reporter line | Number of fragments detected | Integrity | |||
ScaI | BglII | BglII/PciI | |||||
bar | eYFP | bar | eYFP | eYFP | |||
Single locus integrations | 1 | 19[2]-2 | 1 | 1 | 1 | 1 | + |
1 | 19[2]-5 | 1 | 1 | 1 | 1 | + | |
1 | 19[2]-9 | 1 | 1 | 1 | 1 | + | |
1 | 19[2]-11 | 1 | 1 | 1 | 1 | + | |
1 | 19[4]-1 | 1 | 1 | 1 | 1 | + | |
1 | 19[4]-2 | 1 | 1 | 1 | 1 | + | |
2, inverted repeat | 19[2]-10 | 2 | 1 | 1 | 1 | + | |
2, incomplete integration | 19[2]-3 | 1 | 2 | 1 | 1 | ND | |
Multiple locus integrations | 2 | 19[2]-6 | 2 | 2 | 2 | ND | |
2 | 19[2]-7 | 2 | 2 | 2 | 2 | ND | |
3/4 | 19[2]-1 | 4 | 3 | 3 | 3 | ND | |
ND – not determined. |
Table 1: Summary of the DNA blotting data for transgenics isolated after transformation with pDM19.
Critical to generating transgenics with single, intact integrations of a transgene is the choice of the binary vector used. BIBAC family vectors have been used to deliver sequences of interests to many plant species23,24,25,26,27,28. BIBAC vectors, including BIBAC-GW, yield single-copy integrations with high efficiency: the average number of insertions per line is 1.5 to 2, compared to 3 or higher for most commonly used binary vectors5,9,29. As a major improvement compared to other BIBAC vectors, with the BIBAC-GW vectors, the sequences of interest can be easily inserted using Gateway recombination sites12. The modified vectors overcome the general problems of BIBAC vectors when used in conventional cloning strategies: i) a very limited number of unique restriction sites and ii) a low DNA yield. The Gateway recombination sites make BIBAC-GW vectors an attractive alternative to other binary vectors for generating transgenic plants.
Here a series of protocols, from generating BIBAC-GW derivatives containing sequences of interest, to plant transformation, and DNA blot analysis for number and intactness of the transgenic sequences is described. Several of the protocols reported in this paper, Gateway cloning, electroporation of bacteria, and plant transformation, are common practice in many laboratories and can also be carried out with slight modifications. It is important to know that BIBAC-GW is a single-copy vector in E. coli and in A. tumefaciens. Therefore, when isolating DNA, the yield is low; it is recommended to scale up the isolation procedure.
In transgenics carrying multiple T-DNA integrations, introduced transgenes are often subjected to gene silencing1,2,4,30, and should therefore for most applications be avoided. To identify transgenic plants with single, intact integrations, it is recommended to use DNA blot analysis. While methods other than DNA blotting can be used for determining T-DNA copy number and integrity of the T-DNAs in transgenic lines (segregation analysis, TAIL-PCR, quantitative PCR (qPCR), and digital droplet PCR), although labor intensive, DNA blotting is often the method of choice. Segregation analysis is not able to differentiate between multiple and single T-DNA integrations at single loci. TAIL-PCR often under-estimates the copy number, especially if more than one T-DNA integration is present31, and qPCR needs elaborate optimization for reliable results31,32. Digital droplet PCR is a rather precise method for copy number detection if the equipment required is available31. The added benefit of DNA blotting is facile detection of truncated T-DNAs, which is easily missed in all PCR-based techniques.
With DNA blot analysis, the hybridizing fragments on the blot need to be well identifiable in signal and size. Several factors are known to affect the outcome of DNA blotting. In addition to an appropriate selection of restriction enzymes (strategy indicated in Figure 4) and size markers, sufficient DNA of good quality is required. Less than 2 µg of genomic Arabidopsis DNA will not yield well-identifiable fragments. When dealing with bigger genomes, more DNA is required. To obtain sufficient amounts of Arabidopsis DNA, floral tissues or 1-week old seedlings can be used. A batch of seedlings grown on one Petri dish yields 2–8 µg of DNA. In order to avoid DNA degradation during isolation, care should be taken to process the plant material fast. Furthermore, genomic DNA should be resuspended in Tris-EDTA to reduce its degradation by nucleases, and stored at 4 °C rather than -20 °C to prevent DNA nicking due to repeated freezing-thawing cycles. If unsure that all DNA samples are digested completely, it is suggested to rehybridize the DNA blot with a probe recognizing an endogenous, unique genomic region. When selecting probe sequences for identifying transgenic or endogenous sequences, it is crucial to only select unique sequences. To be able to precisely determine the size of hybridized fragments, the positions of DNA gel slots and DNA marker bands should be marked on a transparency (Figure 5B) when visualizing an Ethidium Bromide-stained gel (Figure 5A) on a UV transilluminator. In case the marker sequences do not hybridize with the probe DNA, or hybridization is partial, it is the only way to track down the size of hybridizing fragments.
Once care is taken to achieve good hybridization signal and estimate of the fragment size, the interpretation of the blotting results is straightforward. When using only one restriction enzyme, and hybridizing with different probes detecting either the left or right part of the T-DNA, the number of detected fragments reflects the number of T-DNA insertions. For instance, Figure 7B, C shows the same DNA blot, hybridized with different probes, bar (Figure 7B) and enhanced Yellow Fluorescent Protein (eYFP) (Figure 7C), using the strategy shown in Figure 7A. All lanes, except 4, show an equal number of fragments on both blots: two fragments for line 6 and a single fragment for all other lines. This number of detected fragments is the number of T-DNA insertions.
When the number of fragments detected with probes binding either the left or right part of the T-DNA differs (as for Figure 7B, C, line 4), either incomplete insertions are present, or the T-DNAs have inserted in tandem arrangement. The tandem insertions display non-random fragment length for one of the T-DNA fragments (Figure 4A, right panel), and can be identified by comparing the hybridized fragment size with what is expected based on the restriction strategy. An additional blotting strategy may be needed to confirm the tandem arrangement of the T-DNA insertions. In the sample shown on line 4 (Figure 7B, C), two insertions are arranged in an inverted repeat orientation.
When estimating the intactness of a T-DNA, or part of it, the length of the hybridizing fragment can be calculated based on the restriction strategy. Any deviation from the expected size indicates the presence of an incomplete insertion. For instance, in Figure 7D, in lane 4, a hybridizing fragment migrates at 8 kbp, (instead of the expected 5.5 kbp) indicating an increased fragment size due to the lack of one of the restriction sites.
BIBAC-GW vectors are excellent tools for generating single-copy intact integrations in a number of plant species. The protocol reported here provides a reliable procedure to identify plants with single, intact integrations of a transgene of interest.
The authors have nothing to disclose.
This research is supported by the Dutch Technology Foundation STW (12385), which is part of the Netherlands Organization for Scientific Research (NWO), and which is partly funded by the Ministry of Economic Affairs (OTP Grant 12385 to MS). We thank Carol M. Hamilton (Cornell University, United States) for providing pCH20, the backbone of the BIBAC-GW vectors.
Kanamycin sulphate monohydrate | Duchefa | K0126 | |
Gentamycin sulphate | Duchefa | G0124 | |
Rifampicin | Duchefa | R0146 | |
Tetracycline hydrochloride | Sigma | T-3383 | |
DB3.1 competent cells | Thermo Scientific – Invitrogen | 11782-018 | One Shot ccdB Survival 2 T1R Competent Cells (A10460) by Invitrogen or any other ccdB resistant E. coli strain can be used instead |
DH10B competent cells | Thermo Scientific – Invitrogen | 18290-015 | |
Gateway LR clonase enzyme mix | Thermo Scientific – Invitrogen | 11791-019 | |
tri-Sodium citrate dihydrate | Merck | 106432 | |
Trizma base | Sigma-Aldrich | T1503 | |
EDTA disodium dihydrate | Duchefa | E0511 | |
Proteinase K | Thermo Scientific | EO0491 | |
Bacto tryptone | BD | 211705 | |
Yeast extract | BD | 212750 | |
Sodium chloride | Honeywell Fluka | 13423 | |
Potassium chloride | Merck | 104936 | |
D(+)-Glucose monohydrate | Merck | 108346 | |
Electroporation Cuvettes, 0.1 cm gap | Biorad | 1652089 | |
Electroporator Gene Pulser | BioRad | ||
Magnesium sulfate heptahydrate | Calbiochem | 442613 | |
D(+)-Maltose monohydrate 90% | Acros Organics | 32991 | |
Sucrose | Sigma-Aldrich | 84100 | |
Silwet L-77 | Fisher Scientific | NC0138454 | |
Murashige Skoog medium | Duchefa | M0221 | |
Agar | BD | 214010 | |
Glufosinate-ammonium (Basta) | Bayer | 79391781 | |
Restriction enzymes | NEB | ||
Ethidium Bromide | Bio-Rad | 1610433 | |
Electrophoresis system | Bio-Rad | ||
Sodium hydroxide | Merck | 106498 | |
Hydrochloric acid | Merck | 100316 | |
Blotting nylon membrane Hybond N+ | Sigma Aldrich | 15358 | or GE Healthcare Life Sciences (RPN203B) |
Whatman 3MM Chr blotting paper | GE Healthcare Life Sciences | 3030-931 | |
dNTP | Thermo Fisher | R0181 | |
Acetylated BSA | Sigma-Aldrich | B2518 | |
HEPES | Sigma-Aldrich | H4034 | |
2-Mercaptoethanol | Merck | 805740 | |
Sephadex G-50 Coarse | GE Healthcare Life Sciences | 17004401 | or Sephadex G-50 Medium (17004301) |
Dextran sulfate sodium salt | Sigma-Aldrich | D8906 | |
Sodium Dodecyl Sulfate | US Biological | S5010 | |
Salmon Sperm DNA | Sigma-Aldrich | D7656 | |
Sodium dihydrogen phosphate monohydrate | Merck | 106346 | |
Storage Phosphor screen and casette | GE Healthcare Life Sciences | 28-9564-74 | |
Phosphor imager | GE Healthcare Life Sciences | Typhoon FLA 7000 | |
UV Crosslinker | Stratagene | Stratalinker 1800 | |
cling film (Saran wrap) | Omnilabo | 1090681 | |
Agarose | Thermo Scientific – Invitrogen | 16500 | |
Boric acid | Merck | 100165 | |
DNA marker ‘Blauw’; DNA ladder. | MRC Holland | MCT8070 | |
DNA marker ‘Rood’; DNA ladder | MRC Holland | MCT8080 | |
Hexanucleotide Mix | Roche | 11277081001 | |
Large-Construct Kit | Qiagen | 12462 | |
Heat-sealable polyethylene tubing, clear | various providers | the width of the tubing should be wider than that of blotting membrane | |
Heat sealer | |||
Membrane filter disk | Merck | VSWP02500 | |
Magnesium chloride | Merck | 105833 | |
Hybridization mesh | GE Healthcare Life Sciences | RPN2519 |