The X enopus oocyte expression system

Gordon and co-workers first introduced the Xenopus laevis oocyte as a viable

expression system for messenger RNA (mRNA) injected into the oocyte cytoplasm in 1971. When Xenopus laevis oocytes were injected with 9S mRNA coding for

heterologous haemoglobin and incubated at 19°C, they rapidly and efficiently

synthesised the haemoglobin protein (as revealed by radiolabelling with ^H-histidine; see Gordon et a l, 1971).

However, it was Sumikawa et al (1981) who first showed ûvàX Xenopus laevis oocytes could correctly assemble and express heterologous ion channels: using cat skeletal muscle mRNA, they obtained muscle nAChR. Boulter et al. (1987) were the first group to express a neuronal nAChR in the oocyte by injecting cRNA (rat a3p2). Since these early experiments, the oocyte has become an established expression system used by many electrophysiologists to study various ion channels (Sigel, 1990). ThQ Xenopus oocyte expression system was chosen for the present study because oocytes assemble nAChR efficiently and it is easy to control the proportion o f subunits transfected for complex subunit combinations (for a review see Sivilotti et a l, 2000).

Xenopus laevis oocyte is the precursor o f the egg cell, which leaves the oviduct to become fertilised. Oocytes are stored in the ovaries in the abdominal cavity o f the female Xenopus, from where they can be surgically removed for use in

electrophysiological experiments (for a review see Smart and Krishek 1995). Oocytes are ideally suited for use in electrophysiological experiments due to their large size and ability to efficiently assemble and express exogenous proteins on their cell surface.

Because o f its size, the oocyte is relatively resistant to damage by electrodes and can be injected individually with the genetic material coding for the receptor o f choice.

2.2.1. Xenopus oocyte preparation

Female Xenopus laevis frogs (Blades, UK) were anaesthetised by immersion in neutralised ethyl m-aminobenzoate solution (tricaine, methanesulphonate salt; 0.2 % solution weight/volume; Sigma Chemical Co.), and killed by decapitation, followed by destruction o f the brain and spinal cord (in accordance with Home Office guidelines) before removal o f ovarian lobes. Clumps o f approximately 300 cells freshly removed from the ovarian lobes were dissected in pre-incubation Barth’s solution into groups o f approximately 70 cells, discarding small immature cells that lacked well defined poles and uniform pigmentation. The dissected oocytes were gently shaken (Mini Orbital S05, Stuart Scientific) in collagenase solution (245 collagen digestion units m f ’ in pre­ incubation Barth’s solution, 10-12 oocytes per ml; collagenase type lA, Sigma

Chemical Co) for 65 minutes at 18 ±1° C ; repeatedly rinsed with fresh, syringe filtered Barth’s solution (0.22 pm Millex GP; Millipore, Bedford, USA) and stored at 4° C overnight in sterile tissue culture dishes (Falcon, Becton Dickinson). Collagenase treated oocytes were then manually defolliculated in order to allow cRNA injection (watchmaker forceps, Precista No. 5; Excelta Corporation, Buellton, USA) and the largest cells with well defined poles and uniform pigmentation were chosen for cRNA injection.

2.2.2. nA C hR injection and expression

The oocytes chosen for injection were placed on a nylon mesh (24 mm X 15 mm, with a 1 1 X 1 9 grid and each square o f the grid measuring 1 mm X 1 mm, glued to a small Petri dish 37 mm in diameter) for support and immersed in pre-injection Barth’s solution (see figure 2.2.2.a). Injection pipettes were pulled (Drummond glass # 3-000- 203-G/X, pulled on List Medical L/M-3-PA) and broken to form a staggered tip o f 12 -

16 pm (Narishige MF-830 micro forge). The staggered tip was then fire polished (Narishige MF-830 micro forge) and forged into the shape o f a hypodermic needle, in order to minimise damage to the oocyte membrane. In order to reduce cRNA

degradation by RNAses, cRNA for loading the injection pipettes was stored in ethanol washed and fire polished borosilicate glass (G C l50-7.5; Clark Medical). Injection pipettes were backfilled with mineral oil (M-5904) and loaded from the tip with the appropriate cRNA, freshly defrosted from -80°C and centrifuged (to accumulate the solution at the bottom o f the vial).

cRNA was then injected into the cytoplasm o f the oocyte with a Drummond Nanoject (Drummond Instruments; see figure 2.2.2.a) and confirmed by a visible real time increase in the diameter o f the oocyte (“plumping up”). Upon completion o f the injection, the oocytes were rapidly transferred with a fire polished Pasteur pipette to individual wells in a multiwell plate (24 Multiwell, Falcon, Becton Dickinson), filled with syringe filtered post-injection Barth’s solution (0.22 pm Millex GP; Millipore, Bedford, USA).

The ratio o f a to p was 1:1 for a3p4. When expressing a3p4p3 or a 3 p 4 a5 receptors, a 1:1:20 ratio (a3:p4:p3/a5) was chosen to ensure that most o f the current was carried by

triplet receptors (rather than by pair a3(34, Groot-Kormelink et a l, 1998). The amount o f cRNA to be injected (in 46 nl o f RNAse-free water) for each combination was determined empirically, with the aim o f achieving a maximum ACh-evoked current o f 1.5-2 pA. The maximum amount o f cRNA injected for each combination is described in table 2.4.2.a.

The oocytes were then incubated for approximately 60 hours at 18° C (BDH cooled incubator; BDH). After incubation, oocytes were stored at 4° C until needed for recording. Experiments were carried out at a room temperature o f 18-20° C between 2.5 and 14 days from injection.

Figure 2.2.2.a. The oocyte cRNA injection setup

B

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DAMOtn

F ig ure 2 .2 .2 .a. A sh o w s th e g e n e ra l se t up fo r th e c R N A in je ctio n of oocytes. HS = H am ilto n syrin g e used for disp e n sin g cR N A , DNJ = D ru m m o n d N a n o je ct, M O = sy rin g e fille d w ith m in e ra l oil fo r b a ckfillin g in je ctio n p ip e tte s, NM = nylo n m e s h fo r s u p p o rtin g o o cyte s. B s h o w s the nylon m e sh p ro vid in g s u p p o rt fo r o o cytes im m e rse d in p re -in je c tio n B a rth 's so lutio n, a w a itin g in je ctio n . C sh o w s a m a g n ifie d im ag e

of B.