rpc00069: Coptis japonica 156-S transgenic cell suspension culture

Note

  • Components

    • Domestic delivery: A 50-mL plastic conical centrifuge tube, containing cell suspension

    • Overseas delivery: A 250-mL plastic Erlenmeyer flask, containing cells placed on semi-solid medium

  • Notice

    • Subculture the cells to fresh medium immediately after arrival.

    • Do not store the cell culture in a refrigerator and a freezer.

    • Maintain aseptic conditions of the cell culture, and work in a laminar flow cabinet.

  • Method

    • Culture medium: LS medium, 10 µM NAA, 0.01 µM BAP, pH 5.7 (medium no. 54)

    • Culture conditions: 25°C, dark, 90 rpm

    • Subculture: 21-day intervals

  • Citation of cell line

    When results obtained by using this cell line are published in a scientific journal, it should be cited in the following manner: “Coptis japonica 156-S cell line (rpc00069) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”

Introduction

Coptis japonica 156-S cell line is a transgenic cell line over-expressing Coptis (S)-scoulerine 9-O-methyltransferase (Sato et al. 2001 [1]). The 156-S is a yellow high berberine-producing cell line. The parent cell line 156-1 was established from a petiole-derived root culture of Coptis japonica (Thunb.) Makino var. dissecta (Yatabe) Nakai ex Satake (Yamada and Sato 1981 [3], Sato and Yamada 1984 [2]). The 156-S cells are grown in a Linsmaier and Skoog (LS) medium supplemented with 10 µM 1-naphthaleneacetic acid (NAA) and 0.01 µM 6-benzylaminopurine (BAP), pH 5.7. Our 156-S cell culture has been maintained in the dark at 25°C with rotary shaking at 90 rpm and subcultured at 21-day intervals.

Materials

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

  • MS salt mix

    Murashige and Skoog Plant Salt Mixture, FUJIFILM Wako Pure Chemical Corporation (#392-00591)

  • Sucrose

  • LS_VT

    Chemical

    Concentration (mg/mL)

    Thiamine·HCl

    0.16

    myo-Inositol

    40

  • NAA (1 mM)

    Chemical

    Concentration (mg/mL)

    NAA·K

    0.224

    Potassium 1-naphthylacetate, FUJIFILM Wako Pure Chemical Corporation (#161-04021)

  • BAP (1 mM)

    Chemical

    Concentration (mg/mL)

    6-Benzylaminopurine

    0.225

    Dissolve 6-benzylaminopurine in small volume of KOH (1 N), and fill up with distilled water

  • KOH (1 N)

Glassware

  • Erlenmeyer flask (300 mL), capped with two layers of aluminum foil, sterilized by autoclaving at 121°C for 20 min

Preparation of LS medium (medium no. 54)

  1. Dissolve the following chemicals in approximately 800 mL of distilled water.

    Chemical

    Amount

    MS salt mix

    1 bag (1 L)

    Sucrose

    30 g

  2. Add following stock solutions, and fill up to approximately 950 mL with distilled water.

    Stock solution

    Volume (mL)

    LS_VT

    2.5

    NAA (1 mM)

    10

    BAP (1 mM)

    0.01

  3. Adjust the pH of the solution to 5.7 with KOH (1 N), and fill up to 1 L with distilled water.

  4. Autoclave the medium at 121°C for 20 min.

Methods

  1. Leave to stand a flask containing a 21-day-old culture until the cells settle to the bottom of the flask.

  2. Discard culture medium by decantation.

  3. Wash the cells with a fresh LS medium two times. If the cell amount is too much, discard some cells with the medium.

  4. Add 1.5 times the volume of fresh LS medium to the cells (up to a total volume of approximately 100 mL).

  5. Incubate cell cultures on a rotary shaker at 90 rpm under the dark condition at 25°C.

Notes

  • For domestic customers: We send 156-S cell suspension in a 50-mL disposable conical centrifuge tube. The cells should be transferred to fresh LS medium immediately after arrival. Transfer settled cells to Erlenmeyer flasks containing fresh liquid medium with a pipette.

  • For overseas customers: We send 156-S cells placed on semi-solid LS medium in a 250-mL disposable Erlenmeyer flask. The cells should be transferred to fresh LS medium immediately after arrival. Collect the cells from the semi-solid medium with a spatula and transfer them to Erlenmeyer flasks containing fresh liquid medium.

  • In order to maintain 156-S cell suspension cultures stably, it is essential to transfer an adequate amount of cells to fresh LS medium in every subculture. The amount of cells may vary from one lab to another, because proliferation of 156-S cells is affected by culture conditions, such as a room temperature, rotation speed of a rotary shaker, and aeration condition of the culture.

  • In order to obtain good aeration of a suspension culture, a silicone sponge plug may be used instead of the aluminum foil cap (e.g., cap-type Silicosen; Shin-Etsu Polymer, Tokyo, Japan).

  • Growth of 156-S is very slow. Do not significantly reduce the cell amount in the liquid medium.

Maintenance history

References