Materials and Reagents

Glass capillaries (Drummond Scientific, catalog number: 3-000-203-G/X )
Slide glass
Parafilm M (LMS, catalog number: 94-2542-5 )
Petri dishes (Diameter: 3.5 cm and 9.0 cm, LMS, Japan)
Pipette tips
2 ml Eppendorf tube (Eppendorf, catalog number: 022363344 )
Eppendorf safe-lock tubes,1.5 ml (Eppendorf, catalog number: 0030120086 )
Metal-free tube without lids (DigiTUBEs, GL Sciences, catalog number: 8520-50112 )
Syringe with needle
Polypropylene mesh (0.8 mm, Spectrum, catalog number: 146492 )
DigiFILTER 0.45 μm (SCP SCIENCE, catalog number: 010-500-070 )
Xenopus leavis oocytes treated with collagenase (Kitagawa Institute, hita, Japan)
Note: Protocol of harvesting of ovaries from female Xenopus laevis is available (Shi and Carattino, 2017).
pXBG-ev1 vector (Preston et al., 1992; Figure 2)
Gel extraction kit (Sigma-Aldrich, catalog number: NA1111 )
TE buffer
Ultra pure water produced using the MILLI-Q ADVANTAGE A10 purification system (Millipore)
Note: To prepare samples for ICP-MS, do not use glassware made by borosilicate to avoid contamination of B.
mMESSAGE mMACHINE T3 Transcription Kit (Thermo Fisher Scientific, Invitrogen^TM, catalog number: AM1348)
Mineral oil (NACALAI TESQUE, CAS number: 8020-83-5)
Boric acid (Wako Pure Chemical Industries, catalog number: 029-02191 )
Nitric acid (HNO₃) (For boron determination, Wako Pure Chemical Industries, catalog number: 140-05415 )
Boron standard solution (B 1,000, Wako Pure Chemical Industries, catalog number: 025-16581 )
Modified Barth’s Saline (MBS) buffer with or without Ca²⁺ (see Recipes)
1. Sodium chloride (NaCl, Wako Pure Chemical Industries, catalog number: 191-01665 )
2. Potassium chloride (KCl, Wako Pure Chemical Industries, catalog number: 163-03545 )
3. Sodium bicarbonate (NaHCO₃, NACALAI TESQUE, catalog number: 31212-25 )
4. Tris-HCl (pH 7.6, Sigma-Aldrich, Roche Diagnostics, catalog number: 10708976001 )
5. Calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O, Wako Pure Chemical Industries)
6. Calcium chloride dihydrate (CaCl₂·2H₂O, Wako Pure Chemical Industries, catalog number: 039-00431 )
7. Magnesium sulfate heptahydrate (MgSO₄·7H₂O, Wako Pure Chemical Industries, catalog number: 131-00405 )
8. Sodium penicillin (Wako Pure Chemical Industries, catalog number: 168-23191 )
9. Streptomycin sulfate (Wako Pure Chemical Industries, catalog number: 196-08511 )

Equipment

Dual-Stage Glass Micropipette (NARISHIGE, model: PC-10 )
Confocal laser scanning microscope (Leica Microsystems, model: Leica TCS SP8 )
Fluorescent microscope (Olympus, model: MVX-10 )
Nanoliter injector (Drummond Scientific, model: Nanoject II )
Stereo microscope (Nikon, model: SMZ-2T )
Incubator (Advantec, model: THS030PA )
Heat digestion system (GL Sciences, model: DigiPREP Jr )
ICP-MS (PerkinElmer, model: ELAN DRC-e )
Autosamplers (PerkinElmer, model: ESI )
Ultrapure water system (Merck, model: MILLI-Q^® ADVENTAGE A10 )

Procedure

An outline of procedures to express boric acid channels in oocytes is illustrated in Figure 1.

Figure 1. Procedures to express boric acid channels in oocytes. The GFP-NIP5;1 ORF sequence was subcloned into pXBG-ev1 vector using the BglII site. The vector was linearized by BamHI and used for in vitro cRNA transcription. GFP-NIP5;1 cRNA was injected into oocytes for expression and subsequent boron transport assay.

cRNA synthesis of NIP5;1
1. Subclone ORF sequence of GFP-NIP5;1 in pXBG-ev1 vector (Figure 2) using BglII site.
  Note: The GFP was used to select oocytes expressing NIP5;1 in a later step (Procedure C).
  
  Figure 2. A map of the pXBG-ev1 vector for in vitro transcription (Preston et al., 1992). An ORF sequence of gene of interest can be subcloned between the 5’ untranslated region (UTR) and 3’ UTR using BglII site.
2. Linearize the plasmid by endonuclease such as BamHI. Purify DNA fragment with a gel extraction kit (Sigma-Aldrich) and dilute to be the concentration of 500 ng/µl in TE buffer or nuclease-free water.
3. Run capped transcription reaction and recover cRNA following the manufacturer’s instruction. We use mMESSAGE mMACHINE T3 Transcription Kit (Thermo Fisher Scientific).
  Note: The manufacturer’s guide for cRNA synthesis could be found at https://www.thermofisher.com/order/catalog/product/AM1348.
cRNA injection of NIP5;1
1. Prepare sharp glass capillary by dual-stage glass micropipette puller PC-10 (Figure 3).
  Note: Fix a capillary and set the parameters in PC-10 as follows: No.1 Heater level: 60, No.2 Heater level: 58. Press the ‘start’ button to process automatically.
  
  Figure 3. Preparation of glass capillaries by the puller PC-10. Fixation (A), heating and extension (B) of a glass capillary on PC-10. A glass capillary with sharp tip end (C).
2. Break tip end by the cut-surface of a slide glass under a stereo microscope and check the sharpness of the glass tip under a stereo microscope (Figures 4A-4C).
  Note: Check the tip-end by taking MilliQ water. Usually the glass capillary made by PC-10 cannot be used directly because of the sealed tip-end (Figure 4D).
  
  Figure 4. Examination of the glass capillary. A. The process of breaking tip end by the cut-surface of a glass slide; B. Observation of tip end of glass capillary in stereo microscopy; C. A close-up view of interface of slide and tip end indicated by the white arrowhead; D. Examination of the hole at the tip end of glass capillary by taking water.
3. Fill mineral oil up capillary using a syringe with needle, and equip the capillary on the injector NANOJECT II (Figures 5A-5C).
  
  Figure 5. Filling the glass capillary with mineral oil. Filling the glass capillary with mineral oil by a syringe (A) and setting it on NANOJECT II (B, C). The white circle in B indicates the position of glass capillary on NANOJECT II.
4. Incubate oocytes in MBS with Ca²⁺ buffer (see Recipes) at 18 °C for 6 h prior to injection.
  Note: Select healthy oocytes with clear black and yellow halves for injection (Sigel, 2010).
5. For each cRNA sample, prepare 35 oocytes and inject 50 nl of cRNA solution for one oocyte by NANOJECT II (Figure 6).
  Note: Press ‘empty’ button to push out metal needle to the position at ~0.5 cm from tip-end of glass capillary. Place 4 µl of cRNA on Parafilm (Figure 6B) and take up by pushing ‘fill’ button (Figure 6A). Transfer several oocytes on polypropylene mesh placed in a new Petri dish by 1 ml end cut pipette tips (Figure 6C). Move microinjector close to an oocyte and gently insert glass capillary. Press the ‘inject’ button of NANOJECT II for injection. Detach the glass capillary from the oocyte and sequentially inject next one. Change capillary when you deal with different cRNA.
  
  Figure 6. cRNA injection into oocytes by NANOJECT II. A. Control panel of NANOJECT II; B. Filling a glass capillary with cRNA; C. cRNA injection into oocytes by NANOJECT II.
Incubation of oocytes with boric acid
1. Observe expression of GFP-fused proteins in injected oocytes under a fluorescent stereo microscopy after 1-2 days incubation in MBS with Ca²⁺buffer (see Recipes) at 18 °C (Figures 7A-7C).
2. Image localization of GFP-fused protein in the oocyte by confocal laser scanning microscopy (Figures 7D-7F).
  
  Figure 7. Observation of GFP in oocytes. A-C. Observation of GFP signals in oocytes injected with cRNA by a fluorescent microscope. Oocytes injected with TE buffer (B) and GFP-NIP5;1 cRNA (C), respectively. D. Observation of oocytes by a confocal laser scanning microscopy; E and F. A surface image of oocytes (white arrowheads) (E) and a medial-optical section of oocyte (F) injected with GFP-NIP5;1 cRNA. Scale bars = 1.0 mm (7B, 7C, 7E), 0.5 mm (7F).
3. Collect 7-8 oocytes as one pool in a 2 ml Eppendorf tube using 1 ml end cut pipette tip. Preparing 4 or more replicas is recommended.
4. Remove the MBS buffer and add 1 ml of MBS containing 5 mM boric acid into each tube. Incubate for 30 min at 18 °C.
5. Remove the MBS buffer and wash oocytes for 5 times using 1 ml of fresh MBS buffer without boric acid.
B measurement by ICP-MS
1. Transfer one pool of oocytes into a new metal-free tube without lids (we used DigiTUBEs, GL Sciences). Add 500 µl concentrated HNO₃to digest 20 min, followed by incubation at 100 °C in the DigiPREP Jr equipment (GL Sciences, Tokyo). Shake DigiTUBEs every 2 min to dry the solution completely. Resuspend the sample with 165 µl concentrated HNO₃ and then add ultrapure water up to 5 ml to make 2% HNO₃ solution.
2. Add an appropriate volume of 2% HNO₃ to dilute samples for ICP-MS measurement. We prepared 2% nitric acid containing solution and measure the boron concentration.
  Note: Occasionally, undissolved materials remain in DigiTUBEs. They can be eliminated by DigiFILTER 0.45 μm (GL Sciences). 1 to 5% HNO₃ can be used dependent on ICP-MS instruments.
3. Measure ¹¹B using 0 to 100 ppb total B (natural abundance) as standards by ICP-MS (Figure 8). We used ICP-MS ELAN DRC-e equipped with ESI Autosamplers and software suit (PerkinElmer, Waltham, Massachusetts, USA) We calculated the total B concentration assuming that there is no isotopic discrimination during the transport (¹⁰B:¹¹B = 20:80).
  Note: The guide for ICP-MS operation could be found at http://helpwiki.evergreen.edu/wiki/index.php/PerkinElmer_ELAN_DRC-E_ICP-MS_Manuals.
  
  Figure 8. Linear equation of B standards and intensity measured by ICP-MS. Pre: Measurement of standards prior to measurement of samples. Post: Measurement of standards after measurement of samples.

Data analysis

We calculated the total B content in each oocyte as nmol/oocyte (Table 1). Significantly higher B content in GFP-NIP5;1 expressing oocytes than in TE injected control indicates that GFP-NIP5;1 has boron transport activity (Figure 9).

Table 1. Example of data analysis

Figure 9. Boron content in oocyte after incubation with 5 mM boric acid for 30 min

Recipes

Modified Barth’s Saline (MBS) buffer with or without Ca²⁺(Table 2)
1. High salt stock
  64 g NaCl
  1 g KCl
  2.5 g NaHCO₃
  22.5 g Tris-base
  pH 7.6 by concentrated HCl
  Fill up to 500 ml with MilliQ water and store at 4 °C
2. Ca²⁺ stock
  0.95 g Ca(NO₃)₂·4H₂O
  0.755 g CaCl₂·2H₂O
  Fill up to 100 ml with MilliQ water and store at 4 °C
3. Mg²⁺ stock
  2.5 g MgSO₄·7H₂O
  Fill up to 100 ml with MilliQ water and store at 4 °C
4. Penicillin stock
  Sodium penicillin 10 mg/ml (0.1 g/10 ml)
5. Streptomycin stock
  Streptomycin sulfate 10 mg/ml (0.1 g/10 ml)
  
  Table 2. Preparation of MBS buffer with or without Ca²⁺

Acknowledgments

We thank Jian Feng Ma, Naoki Yamaji (Okayama University), Toshihiro Watanabe and Tomoko Shimizu (Hokkaido University) for their help in fluorescent confocal microscopy and ICP-MS measurement. This work was supported by the NEXT program (GS001) and the Grant-in-Aid for Young Scientists (A) (26712007) from the Japan Society for the Promotion of Science, the Young Investigators Grant from the Human Frontier Science Program (RGY0090/2011), and a research grant from the Naito foundation to J.T. The authors declare no conflicts of interest or competing interests.

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