20 Expression Cloning with Xenopus Oocytes
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6. The oocytes are sorted for morphologically intact, healthy looking stage
V-VI oocytes. The oocytes are subsequently maintained in MBS at 18 oc
(kept in the dark).
7. U sually on the next day, healthy looking stage V-VI oocytes are injected
with either 50 nl water (control) or mRNA (at concentrations varying
from 0 to 1 )lg/)ll), using a semiautomatic injector. Oocytes are then
kept at 18°C in MBS for 1 to 7 days, with changes of MBS solution every
1-2 days.
Protein expression (transport) assay
1. Oocytes (7-10 oocytes perindividual data point) are first washed for 1-2
minutes in solution A. This solution is then replaced by 100 J..Ll of solution
A supplemented with the desired concentration of substrate ( eg. 1 mM
K2S04), at the desired specific activity for the isotope. The specific activities for the different substrates used ( eg. 35 Soi-) are usually between 2040 )..LCi/ml. Uptakes are routinely performed in the presence of sodium
(100 mM NaCl) or in its absence (100 mM choline chloride) and incubated in a water bathat 25°C for various times (1-60 min.).
2. After the incubation, the uptake solution is removed and the oocytes are
washed 3 times with 3 ml of ice cold stop solution (solution A).
3. Each single oocyte is then placed into a separate scintillation vial, dissolved in 200 J..Ll of 1% SDS (allowed to shake vigorously for 45 min at
room temp).
4. When the ooyctes have completely dissolved, they are supplemented by 2
ml scintillation fluid (Emulsifier Safe, Kontron). Isotopic uptake (transport assay) is measured by liquid scintillation spectrometry (normally
counted for 2 min/vial).
Construction and screening of a cDNA expression library
1. Using a functionally positive unfractionated or size-fractionated mRNA
as starting material, a directional cDNA library is constructed using a
commercial kit, following the manufacturers protocol (in our case the
SuperScript plasmid system, pSPORT-1 vector, Gibco/BRL; SuperScript
reverse transcriptase having no RNase H activity). The total library,
usually in the range of 10 5 -10 6 clones, is subdivided into pools of
313
6. The oocytes are sorted for morphologically intact, healthy looking stage
V-VI oocytes. The oocytes are subsequently maintained in MBS at 18 oc
(kept in the dark).
7. U sually on the next day, healthy looking stage V-VI oocytes are injected
with either 50 nl water (control) or mRNA (at concentrations varying
from 0 to 1 )lg/)ll), using a semiautomatic injector. Oocytes are then
kept at 18°C in MBS for 1 to 7 days, with changes of MBS solution every
1-2 days.
Protein expression (transport) assay
1. Oocytes (7-10 oocytes perindividual data point) are first washed for 1-2
minutes in solution A. This solution is then replaced by 100 J..Ll of solution
A supplemented with the desired concentration of substrate ( eg. 1 mM
K2S04), at the desired specific activity for the isotope. The specific activities for the different substrates used ( eg. 35 Soi-) are usually between 2040 )..LCi/ml. Uptakes are routinely performed in the presence of sodium
(100 mM NaCl) or in its absence (100 mM choline chloride) and incubated in a water bathat 25°C for various times (1-60 min.).
2. After the incubation, the uptake solution is removed and the oocytes are
washed 3 times with 3 ml of ice cold stop solution (solution A).
3. Each single oocyte is then placed into a separate scintillation vial, dissolved in 200 J..Ll of 1% SDS (allowed to shake vigorously for 45 min at
room temp).
4. When the ooyctes have completely dissolved, they are supplemented by 2
ml scintillation fluid (Emulsifier Safe, Kontron). Isotopic uptake (transport assay) is measured by liquid scintillation spectrometry (normally
counted for 2 min/vial).
Construction and screening of a cDNA expression library
1. Using a functionally positive unfractionated or size-fractionated mRNA
as starting material, a directional cDNA library is constructed using a
commercial kit, following the manufacturers protocol (in our case the
SuperScript plasmid system, pSPORT-1 vector, Gibco/BRL; SuperScript
reverse transcriptase having no RNase H activity). The total library,
usually in the range of 10 5 -10 6 clones, is subdivided into pools of