Efficient transgenesis mediated by pigmentation rescue in zebrafish

Abstract

The zebrafish represents a revolutionary tool in large-scale genetic and small-molecule screens for gene and drug discovery. Transgenic zebrafish are often utilized in these screens. Many transgenic fish lines are maintained in the heterozygous state due to the lethality associated with homozygosity; thus, their progeny must be sorted to ensure a population expressing the transgene of interest for use in screens. Sorting transgenic embryos under a fluorescence microscope is very labor-intensive and demands fine-tuned motor skills. Here we report an efficient transgenic method of utilizing pigmentation rescue of nacre mutant fish for accurate naked-eye identification of both mosaic founders and stable transgenic zebrafish. This was accomplished by co-injecting two constructs with the I-SceI meganuclease enzyme into pigmentless nacre embryos: I-SceI-mitfa:mitfa-I-SceI to rescue the pigmentation and I-SceI-zpromoter:gene-of-interest-I-SceI to express the gene of interest under a zebrafish promoter (zpromoter). Pigmentation rescue reliably predicted transgene integration. Compared with other transgenic techniques, our approach significantly increases the overall percentage of founders and facilitates accurate naked-eye identification of stable transgenic fish, greatly reducing laborious fluorescence microscope sorting and PCR genotyping. Thus, this approach is ideal for generating transgenic fish for large-scale screens.

The zebrafish (Danio rerio) is an ideal organism to study vertebrate development and model human disease due to its high fecundity, rapid ex utero development, and optical transparency, as well as the ability to fluorescently label specific lineages of interest (1). Transgenic zebrafish are often used as tools in high-throughput screens to identify lead compounds, novel genes, and pathways that modify a particular phenotype in development or disease (1,2). In many cases, homozygous transgenic fish cannot be maintained due to the adverse effects of elevated expression of the transgene on zebrafish development and fertility. Therefore, before being utilized in screens, each transgenic embryo from heterozygous outcrosses must be sorted via fluorescence microscopy. This method is labor-intensive, time-consuming, and reliant on robust fluorescent protein expression. Hence, it is important to develop an efficient strategy that enables easy identification of transgenic fish without the need for laborious fluorescence microscopy or conventional PCR genotyping.

Over the past two decades, several transgenic techniques have been developed for the zebrafish system, including viral-mediated transgenesis and the introduction of foreign DNA by microinjection, nuclear transfer, and embryonic cell and tissue culture techniques (3–10). Of these strategies, microinjection of plasmid DNA directly into fertilized eggs has become the preferred technique (6–8). The conventional microinjection technique has a poor efficiency of transgene integration due to the use of linearized plasmid DNA, which favors the formation of extrachromosomal elements. The rate of germline integration for this transgenic method is ~0.5%–5%. Additionally, transgenes are often integrated as concatemers and, thus, are frequently methylated and silenced in future generations (8,11,12). Modifications to this technique led to the development of newer transgenic methods in zebrafish, such as the transposon-mediated and I-SceI meganuclease-mediated approaches (13–18).

The I-SceI meganuclease recognizes a unique 18-bp sequence that is not present in the zebrafish genome and promotes transgenesis by cleavage of two I-SceI recognition sequences flanking the transgenes of interest (15). I-SceI meganuclease-mediated transgenesis results in mosaic expression of the transgene in over 30% of F0 fish and germline integration in 10%–20% of F0 fish (14,15). This increase in the rate of germline integration is a significant improvement over the conventional technique of microinjection of linearized DNA. To facilitate the identification of fish expressing transgenes, the transgenes of interest are often fused with genes expressing fluorescent proteins or co-injected with fluorescent reporter constructs (19). These techniques allow for relatively straightforward identification of F0 founder and stably integrated fish via fluorescence microscope sorting.

Our approach modifies the existing I-SceI meganuclease method by applying the pigmentation rescue of nac^w2 (nacre) mutants as a visible marker of transgene integration and thus eliminating the need for fluorescence microscope sorting. The nacre mutant fish harbor a point mutation in the gene encoding microphthalmia-associated transcription factor a (mitfa), which is required for specification and survival of melanocytes (20). Due to the mitfa mutation, the nacre mutants lack melanophores throughout development, leading to the absence of the four horizontal melanophore stripes that are present in wild-type fish (20). Despite lacking melanophores, nacre fish develop and breed normally (20). In our studies, two I-SceI-containing constructs were co-injected into one-cell-stage nacre mutant embryos: I-SceI-mitfa:mitfa-I-SceI to rescue pigmentation loss and I-SceI-rag2:gene-of-interest-I-SceI to drive the expression of the gene of interest by a tissue-specific promoter (rag2). We found that the pigmentation rescue in these mutants was a faithful predictor of germline integration of the transgenes of interest, allowing us to select for founder fish via phenotype and identify stable transgenic fish with the naked eye. Our strategy eliminates the need for fluorescence microscope sorting and greatly reduces labor as well as the facility space required for screening and maintaining transgenic fish.

Materials and methods

Zebrafish husbandry

Fish used in this study were maintained on a 14 h/10 h light/dark cycle. Zebrafish husbandry was performed as described in the zebrafish facility at the Dana-Farber Cancer Institute (DFCI), according to the IACUC-approved protocol (under a subcontract between the Trustees of Boston University and DFCI) (21).

Plasmid constructs

All plasmids used for this study, with the exception of I-SceI-mitfa:mitfa-I-SceI, were generated by inserting the coding region of the genes of interest under a zebrafish lymphocyte-specific promoter, rag2. The vector used for subcloning was I-SceI-rag2:EGFP-mMyc-I-SceI modified from the rag2:GFP plasmid (a kind gift from Shuo Lin, University of California at Los Angeles) (22). To insert the gene of interest into this backbone vector, the EGFP-mMyc fragment was excised by specific restriction enzymes (see Supplementary Table S1 for details). The promoter and coding region of these genes were flanked by I-SceI meganuclease recognition sites at the 5′ and 3′ ends to facilitate the integration of transgenes into the chromosome (12). The constructs generated for this research include: I-SceI-mitfa:mitfa-I-SceI, I-SceI-rag2:EGFPI-SceI, I-SceI-rag2:mCherry-I-SceI, I-SceI-rag2:mCherry-bcl-2-I-SceI, I-SceI-rag2:EGFP-I-SceI, I-SceI-rag2:EGFP-bclxL-I-SceI, I-SceI-rag2:mCherry-bad-I-SceI, I-SceI-rag2:mCherry-badL99A-I-SceI, and I-SceI-rag2:MYC-ER-I-SceI.

4-hydroxytamoxifen treatment and T cell acute lymphoblastic leukemia (T-ALL) monitoring

4-hydroxytamoxifen (4HT) (Sigma, St. Louis, MO) was administered at a concentration of 129 nM to Tg(rag2:MYC-ER;lck:EGFP) nacre embryos at 5 days post-fertilization (dpf) (23) and was replaced in fresh fish water once a week.

Fish genotyping

DNA was extracted from tail-fin clips or individual embryos as described previously (24). PCR was performed using the gene-specific primers listed in Supplementary Table S3.

Transgene integration site mapping

Genomic DNA was extracted as described above and cleaned up through phenol–chloroform extraction. DNA was subsequently digested with BglII, EcoRI, HindIII, or XbaI restriction enzyme (New England BioLabs, Ipswich, MA). Nested PCR was performed using each digested DNA as template, as described (25). Distinct PCR bands were excised, gel-purified, and sequenced using a gene-specific primer. Nucleotide BLAST search and pairwise sequence comparison were performed to identify the integration site or neighboring transgenes. All primers are listed in Supplementary Table S4.

γ-Irradiation of fish

Adult Tg(rag2:mCherry-bcl2) and Tg(rag2:mCherry) fish were treated with whole-body γ-irradiation (20 Gy from a ¹³⁷Cs source). The fish were imaged prior to and 4 days after irradiation treatment to assess the expression of thymus fluorescence.

Generation of transgenic fish

A microinjection apparatus driven by gas was used to inject nacre mutant embryos at the one-cell-stage with 1 nL of construct mixture containing 1× I-SceI buffer, 0.3 U/μL I-SceI meganuclease enzyme (New England BioLabs), I-SceI-rag2:gene-of-interest-I-SceI (33.3 ng/μL; except for I-SceI-rag2:MYC-ER-I-SceI) and I-SceI-mitfa:mitfa-I-SceI (6.67 ng/μL). To generate Tg(rag2:MYC-ER) fish, I-SceI-rag2-MYC-ER-I-SceI (25 ng/μL) and I-SceI-mitfa:mitfa-I-SceI (5 ng /μL) were co-injected instead.

Epifluorescence microscopy

Adult zebrafish were anesthetized with 0.003% MS222 (Sigma) and photographed on a dissecting microscope (Leica Microsystems, Buffalo Grove, IL). Larvae were similarly anesthetized and photographed using a Zeiss (Thornwood, NY) Axioskop microscope. All images were processed using Adobe Photoshop.

Results and discussion

The nacre mutants harbor a point mutation in the mitfa gene, which results in the formation of a premature stop codon that causes pigmentation loss throughout fish development (20). Despite lacking pigmented skin cells, reproductive capacity and organ development are unaffected in nacre mutants (20). Pigmentation loss in nacre mutants can be rescued by microinjecting wild-type mitfa:mitfa DNA into one-cell-stage embryos (20). Moreover, co-injected constructs are often co-integrated into the genome (26). Thus, we reasoned that the pigmentation rescue can be utilized as a visual marker for the integration of the transgene of interest. Our experimental design was to co-inject two types of I-SceI-containing constructs into nacre pigmentless embryos: I-SceI-mitfa:mitfa-I-SceI to rescue the pigmentation loss and I-SceI-zpromoter:gene-of-interest-I-SceI to express the transgene of interest (Figure 1, A and B).

Figure 1. Generation of transgenic zebrafish via pigmentation rescue.

(A) Schematic of the two constructs that are used in this study. (B) An overview of the transgenic strategy mediated by pigmentation rescue.

To test our prediction, we co-injected the I-SceI-mitfa:mitfa-I-SceI and I-SceI-rag2:MYC-ER-I-SceI constructs into nacre embryos (Figure 2A). As expected, this led to pigmentation rescue in over 50% of the nacre fish (n > 200) (Figure 2, C and D; data not shown). The pigmentation rescue was discernible as early as 3 dpf and remained throughout the lifespan of the fish (Figure 2, C–E; data not shown). Importantly, the co-injection of I-SceI-mitfa:mitfa-I-SceI with I-SceI-rag2:MYC-ER-I-SceI did not affect fish viability, development, or fertility (n > 200) (Figure 2, C–E; data not shown). To determine whether pigmentation rescue predicts the germline integration of the transgene of interest, we separated the F0 founder fish according to the degree of pigmentation rescue, ranging from complete to no rescue (Table 1; Figure 2, B–D; data not shown). We then outcrossed each of these fish to nacre homozygous adults and determined the percentage of pigmented F1 embryos (Figure 1B and Table 1). The F0 founder exhibiting broader pigmentation rescue produced a significantly higher percentage of stable F1 transgenic progeny, as confirmed by MYC-ER PCR (7.46% ± 2.64% for medium-degree pigmented F0 versus 2.57% ± 3.31% for the lightly pigmented F0 fish; n = 3 per group; P = 0.04) (Table 1; Figure 2, C–E). However, pigmentless F0 fish failed to generate pigmented F1 progeny (n = 3; 0% for pigmentless F0 versus 7.46% ± 2.64% for medium-degree pigmented F0; P = 0.045) (Table 1, Figure 2, B and F). Consistent with the absence of pigmentation rescue, conventional DNA extraction and PCR genotyping confirmed no integration of the Tg(rag2:MYC-ER) transgene in pigmentless F1 fish (n > 100) (Figure 2, B, F, and G; Supplementary Table S3; data not shown). These data support the hypothesis that pigmentation rescue in F0 founder fish significantly correlates with germline integration of a transgene. Thus, based on pigmentation rescue, we can easily identify F0 founder fish with germline integration, eliminating the labor required to screen pigmentless fish.

Figure 2. Generation of the Tg(rag2:MYC-ER) line using the pigmentation rescue approach.

Co-injection of (A) I-SceI-rag2:MYC-ER-I-SceI and I-SceI-mitfa:mitfa-I-SceI constructs together with the I-SceI meganuclease into nacre one-cell-stage embryos showed different levels of rescue in F0 adults: (B) no rescue, (C) incomplete rescue with mosaic pigmentation, and (D) complete rescue. (E–F) F1 progeny from the outcross of a pigmented F0 adult to nacre: 3-days post-fertilization (dpf) F1 with pigmentation rescue (E) or lack of pigmentation rescue (F). (G) PCR confirmation that pigmented F1 progeny harbor the Tg(rag2:MYC-ER) transgene. (H–I) Tumor development as visualized by enhanced green fluorescent protein (EGFP) expression in a Tg(rag2:MYC-ER;lck:EGFP) fish (57 dpf) (I) upon 4-hydroxytamoxifen (4HT) treatment, compared with a control fish [Tg(rag2:EGFP)] (50 dpf) (H). Scale bars: B–D = 2 mM, E–F = 500 mm, and H–I = 1 mM.

Table 1.

Tg(rag2:MYC-ER) founder fish are efficiently identified via their pigmentation rescue phenotype

F0 Fish	Pigmentation rescue	Percentage of germline progeny (n)
1	Complete (100% rescue)	61% (n = 18)
2	Medium (~55% rescue)	9.8% (n = 51)
3	Medium (~52% rescue)	8.3% (n = 72)
4	Medium (~50% rescue)	4.3% (n = 47)
5	Light (40% rescue)	6.3% (n = 32)
6	Light (<30% rescue)	1.4% (n = 69)
7	Light (<10% rescue)	0% (n = 83)
8	None	0% (n = 70)
9	None	0% (n = 55)
10	None	0% (n = 72)

We therefore sacrificed the pigmentless F0 fish and only selected the pigmented F0 fish to generate F1 progeny with germline transgene integration (Figure 1B). This method enables rapid naked-eye identification of stable transgenic fish, bypassing the need for laborious fluorescence sorting and conventional genotyping. Using this improved method, a single technician in our laboratory was able to generate 11 founder fish and obtain their stable F1 progeny in less than 4 months, a drastic improvement over previous approaches (Table 2). Moreover, this strategy will enable novice researchers to generate and identify stable transgenic fish based on pigmentation rescue, as only one or two pigmented F0 fish were needed to identify the germline founder (Supplementary Table S2).

Table 2.

Comparison of the pigmentation rescue approach with other transgenic methods

Method	Germline integration (%)	Fish tanks needed	Mating setup	Genotyping method utilized
Linearized-DNA injection (3,7–12)	0.5%–5%	200–300	200–300 pairs	Fluorescence scope or fin-clip
Meganuclease-mediated (14,15)	10%–20%	5–10	5–10 pairs	Fluorescence scope or fin-clip
Fluorescent reporter co-injection (19)	20%–50%	2 –10	2 –10 pairs	Fluorescence scope or fin-clip
Tol2-mediated (17)	10%–50%	2 –10	2 –10 pairs	Fluorescence scope or fin-clip
Pigmentation rescue	88.7% ± 15.7%	2–2	1–2 pairs	Visualization of pigmentation rescue with the naked eye

To determine whether pigmentation rescue can accurately predict the presence of the transgene of interest in the stable transgenic fish, we genotyped the F1 progeny of the Tg(rag2:MYC-ER) founder fish that were outcrossed to nacre mutants. The rag2:MYC-ER transgene was present in all pigmented F1 progeny but in none of the pigmentless F1 siblings (n = 5 per group) (Figure 2, E–G). Therefore, our data indicate that the transgene of interest always co-integrates into the same cell with the mitfa rescue construct (n > 500) (Figure 2G). In total, we generated seven stable transgenic lines using this method (Supplementary Table S2): Tg(rag2:EGFP), Tg(rag2:mCherry), Tg(rag2:mCherry-bcl-2), Tg(rag2:EGFP-bclxL), Tg(rag2:mCherry-bad), Tg(rag2:mCherry-badL99A), and Tg(rag2:MYC-ER). To determine the integration status of the gene of interest transgenic construct and the pigmentation rescue construct, we utilized a restriction-enzyme-PCR-based technique to determine the sequences adjacent to the transgenic construct (25). Our data show that both I-SceI-rag2:MYC-ER-I-SceI and I-SceI-rag2:mCherry-I-SceI constructs co-integrated with the I-SceI-mitfa:mitfa-I-SceI construct as concatemers (Supplementary Figure S1). Consistent with their co-integration status, we observed a Mendelian segregation ratio of transgenes in all of the aforementioned transgenic lines throughout 5 generations (~50% transgene-positive; n > 100 fish per line). Furthermore, we were able to verify the perfect correlation of the transgene presence with pigmentation rescue by PCR-genotyping and fluorescence microscopy examination in four additional transgenic lines generated by the same approach. For instance, the pigmented adult Tg(rag2:mCherry) and Tg(rag2:mCherry-bcl-2) fish expressed robust levels of red fluorescence (mCherry or mCherry-bcl-2) in lymphocytes (n > 20) (Figure 3, A and C), while their pigmentless siblings did not (n > 100) (data not shown). We also observed a similar trend of pigmentation rescue with simultaneous expression of thymus fluorescence in 5-dpf Tg(rag2:mCherry), Tg(rag2:mCherry-bcl-2), Tg(rag2:EGFP), and Tg(rag2:EGFP-bclxL) embryos (n > 100 per transgenic line) (Supplementary Figure S1, A–D). We monitored the pigmented transgenic fish for 10 generations in Tg(rag2:MYC-ER) fish and for 5 generations in the other 6 transgenic lines. Indeed, pigmentation rescue always correlated with the presence of the transgene of interest throughout the generations (data not shown).

Figure 3. mCherry-bcl-2 expression is capable of protecting thymocytes from radiation-induced apoptosis in Tg(rag2:mCherry-bcl-2) fish.

Fluorescence microscopy analysis revealed that mCherrry-expressing T cells were present in the thymus of 3-month-old Tg(rag2:mCherry-bcl-2) transgenic fish without γ-irradiation (IR) (C) or 4 days after IR (20 Gy) (D), while T cells of the Tg(rag2:mCherry) control fish were present in the thymus before IR (A) but disappeared 4 days after IR (20 Gy) (B). Location of the thymus (T) is denoted by arrows. Scale bars: A–D = 1 mM.

To determine whether mitfa expression from the I-SceI-mitfa:mitfa-I-SceI construct impaired fish development, we monitored these seven aforementioned transgenic lines and compared their development to non-transgenic wild-type fish. We found that each of the transgenic lines was viable and fertile, and did not exhibit morphological differences relative to their wild-type siblings. To determine whether transgenic fish generated by our method were functional, we subjected the Tg(rag2:mCherry-bcl-2) and control Tg(rag2:mCherry) fish to ionizing radiation (IR) and monitored thymus fluorescence expression before and after radiation. Due to T cell apoptosis induced by IR, thymus fluorescence in Tg(rag2:mCherry) fish disappeared 4 days post-IR treatment (n > 4) (Figure 3B). However, thymus fluorescence remained in Tg(rag2:mCherry-bcl-2) fish, indicating that the mCherry-bcl-2-expressing thymocytes are resistant to IR-induced apoptosis (n > 4) (Figure 3D). These results obtained from three independent experiments are consistent with a previous report, in which the thymus of Tg(rag2:EGFP-bcl-2) fish generated by the conventional method evaded apoptosis induced by either dexamethasone or IR treatment (27). Furthermore, in agreement with this previous report, we also observed that the thymuses in 5-dpf Tg(rag2:mCherry-bcl-2) embryos were enlarged (Supplementary Figure S2C) compared with those in control Tg(rag2:mCherry) embryos (Supplementary Figure S2A). In comparison to 5-dpf Tg(rag2:EGFP) embryos (Supplementary Figure S2B), we also observed similar thymus enlargement in Tg(rag2:EGFP-bclxL) embryos that expressed the anti-apoptotic bclxL gene in lymphocytes (Supplementary Figure S2D).

MYC overexpression driven by the rag2 promoter promotes rapid T-ALL development in zebrafish (28). Indeed, T-ALL rapidly developed in the Tg(rag2:MYC-ER) line upon tamoxifen treatment (n > 100 per group) (Table 1 and Figure 2I) (23). We crossed the Tg(rag2:MYC-ER) fish to the Tg(lck:EGFP) fish to facilitate the monitoring of tumor development by fluorescence microscopy. By 15 weeks of age, only 10% of the Tg(rag2:MYC-ER;lck:EGFP) fish developed T-ALL in the absence of 4HT treatment (n = 50), while 100% of Tg(rag2:MYC-ER;lck:EGFP) fish treated with 4HT developed T-ALL. Compared with the control Tg(rag2:EGFP) fish (Figure 2H), EGFP-labeled lymphoblasts in Tg(rag2:MYC-ER;lck:EGFP) fish escaped the thymus boundary and widely infiltrated into the head and trunk by 60 dpf (Figure 2I). These results demonstrate that the pigmentation-mediated technique is capable of generating transgenic lines, in which transgene expression and tumor induction can be conditionally regulated.

Among the zebrafish transgenic techniques developed over the last two decades (Table 2), the fluorescent reporter co-injection strategy and Tol2-mediated transgenic approaches attain relatively high rates of transgene integration (20%–50% and 10%–50%, respectively) (Table 2). However, in order to genotype fish with transgene expression, researchers must rely on the labor-intensive methods of fluorescence microscopy and PCR amplification of fish genomic DNA.

Here, we developed a strategy to efficiently and reliably identify both transgenic founder and stable transgenic fish. Based on the number of pigmented fish screened, we can significantly enrich founder fish up to 88.7 ± 15.7% (Table 2 and Supplementary Table S2). This enrichment is likely associated with the fact that we intentionally biased the ratio of the rescue construct to the transgenic plasmid (1:5); thus it is highly likely that fish with pigmentation rescue will harbor the transgene of interest. Our pigmentation-mediated method significantly decreases the number of F0 fish screened for the identification of germline founders. On average, only one or two pigmented F0 fish are screened to identify germline founders (Supplementary Table S2), thus greatly reducing the labor required and number of tanks needed. Pigmented cells on the surface of the skin are easily discernible by the naked eye, whereas fluorescent reporter–mediated transgenesis requires careful microscopic evaluation. Due to the co-integration of the mitfa rescue construct with the transgene of interest, pigmentation rescue serves as a faithful marker for the presence of the transgene of interest in stable transgenic fish and their progeny. These features minimize or even eliminate the need for fluorescence microscopy and laborious genotyping procedures such as fin-clipping, DNA extraction, PCR, and gel electrophoresis.

In addition to the improved rate of transgenesis, the stable transgenic fish developed by our I-SceI meganuclease method exhibit Mendelian segregation of the transgene, suggesting a single-site integration event (12). We used the rag2 promoter to drive the expression of genes of interest. However, other promoters can also be used and should be equally effective in driving transgene expression with this strategy. Examples of transgenic lines generated include the MYCN transgenic fish, in which the dopamine-β-hydroxylase (DβH) promoter was used to drive the expression of MYCN to study neuroblastoma development (29). A recent study has demonstrated that 3 constructs can co-integrate together at a rate of 80% (26). Thus, three I-SceI–containing constructs can be co-injected to express mitfa, a transgene of interest, and a fluorescent reporter gene. Of course, researchers should consider that transgenic fish generated by this method should be maintained in a nacre pigmentless fish background in order to apply pigmentation rescue as a read-out for the presence of the transgene. Once transgenic fish are crossed to pigmented fish, pigmentation rescue can no longer be used as an indicator for transgene integration. Additionally, pigmentation rescue associated with the transgene may limit imaging properties if optical transparency is required. This limitation can be overcome by applying this technique in the context of Tol2-mediated transgenesis because transgenes tend to integrate into different chromosomes, allowing the segregation of the pigmentation rescue construct and transgene of interest through outcrossing in later generations. If one wishes to use pigmentation rescue as a read-out for transgene integration throughout the generations, the rescuing cassette and the experimental construct should be contained in the same plasmid.

Our pigmentation-mediated approach provides a significantly improved strategy for the generation of zebrafish transgenic lines. Due to the ease of screening and sorting fish with the naked eye based on pigmentation rescue, this strategy greatly reduces the labor and space required to generate and maintain transgenic lines that are suitable tools for small molecule and/or genetic screens.

METHOD SUMMARY.

Our new method combines pigmentation rescue of nacre mutants with the I-SceI meganuclease transgenesis strategy. Nacre mutant embryos were co-injected with I-SceI meganuclease and two constructs flanked by I-SceI recognition sites: I-SceI-mitfa:mitfa-I-SceI to rescue the pigmentation defect and I-SceI-zpromoter:gene-of-interest-I-SceI to express the transgene of interest under a zebrafish promoter. Pigmentation rescue reliably predicted germline integration in mosaic founder fish and marked the presence of the transgene of interest in stable transgenic fish.