20 Expression Cloning with Xenopus Oocytes
317
Xenopus laevis:
Isolation of stage VNI oocytes
::
Q
~
ü
"
Q
II
W11ter ~NA A
"
c
111
IV
Tissue of interest:
mRNA isolation
fJ
/"'00
~
VVVVVvvv
13A
t3 8
Fig. 1. Schematic outline of the cloning strategy, using functional expression in Xenopus laevis oocytes. The right panel shows the different cloning steps graphically. The left panel represents the assay with the expected results at the corresponding stage of the project. The
strategy in brief: 1) Isolation of total poly (A)+ RNA from rabbit renal cortex followed by
injection and transport assay (I) in oocytes. 2) Fractionation of the mRNA, injection of
the fractions and transport assay (II) . 3) Construction of a cDNA library from rabbit renal
cortex mRNA. Division of the library into different pools of clones. Synthesis of in vitro transcribed mRNA, injection and transport assay (III). 4) Subdivision of the pool(s) as described
above down to one single clone (IV).
References
Bertran, J, Werner, A, Moore, ML, Stange, G, Markovich, D, Biber, J, Testar, X, Zorzano,
A, Palacin, M and Murer, H (1992) Expression-cloning of a cDNA from rabbit kidney
cortex that induces a single transport system for cystine, dibasic and neutral amino
acids. Proc Natl Acad Sei USA 89: 5601 -5605
Bissig, M, Hagenbuch, B, Stieger, B, Koller, T and Meier, PJ (1993) Functional expression
cloning of the canalicular sulfate transport system of rat hepatocytes. Submitted to: J
Bio! Chem
Guastella, J, Nelson, N, Nelson, H, Czyzyk, L, Keynan, S, Miede!, MC, Davidson, N, Lester,
HA and Kanner, BI (1990) Cloning and expression of a rat brain GABA transporter.
Science 249: 1303-1306
Gurdon, JB, Lane, CD, Woodland, HR and Marbaix, G (1971) Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Nature 233:
177-182
I : mRNA-expression
II: mRNA
slze·seleetionlex.pression
111: cONA·synlhesls.
size-selec tlon,
cONA-IIba ry,
box SCreening
IV: isolation or
single·c:lone
317
Xenopus laevis:
Isolation of stage VNI oocytes
::
Q
~
ü
"
Q
II
W11ter ~NA A
"
c
111
IV
Tissue of interest:
mRNA isolation
fJ
/"'00
~
VVVVVvvv
13A
t3 8
Fig. 1. Schematic outline of the cloning strategy, using functional expression in Xenopus laevis oocytes. The right panel shows the different cloning steps graphically. The left panel represents the assay with the expected results at the corresponding stage of the project. The
strategy in brief: 1) Isolation of total poly (A)+ RNA from rabbit renal cortex followed by
injection and transport assay (I) in oocytes. 2) Fractionation of the mRNA, injection of
the fractions and transport assay (II) . 3) Construction of a cDNA library from rabbit renal
cortex mRNA. Division of the library into different pools of clones. Synthesis of in vitro transcribed mRNA, injection and transport assay (III). 4) Subdivision of the pool(s) as described
above down to one single clone (IV).
References
Bertran, J, Werner, A, Moore, ML, Stange, G, Markovich, D, Biber, J, Testar, X, Zorzano,
A, Palacin, M and Murer, H (1992) Expression-cloning of a cDNA from rabbit kidney
cortex that induces a single transport system for cystine, dibasic and neutral amino
acids. Proc Natl Acad Sei USA 89: 5601 -5605
Bissig, M, Hagenbuch, B, Stieger, B, Koller, T and Meier, PJ (1993) Functional expression
cloning of the canalicular sulfate transport system of rat hepatocytes. Submitted to: J
Bio! Chem
Guastella, J, Nelson, N, Nelson, H, Czyzyk, L, Keynan, S, Miede!, MC, Davidson, N, Lester,
HA and Kanner, BI (1990) Cloning and expression of a rat brain GABA transporter.
Science 249: 1303-1306
Gurdon, JB, Lane, CD, Woodland, HR and Marbaix, G (1971) Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Nature 233:
177-182
I : mRNA-expression
II: mRNA
slze·seleetionlex.pression
111: cONA·synlhesls.
size-selec tlon,
cONA-IIba ry,
box SCreening
IV: isolation or
single·c:lone