312
DANIEL MARKOVICH, ANDREAS WERNER AND HEINI MURER
RN Aaseinhibitor (RNA guard Pharmacia); 50 units T3 or T7 RNA polymerase (Promega).
• phenol:chloroform:isoamylalcohol (25:24: 1)
• collagenase A ( or D ), low trypsin activity (Boehringer, Mannheim; 2 mg/
ml)
• mRNA (of choice)
• Restrietion enzyme cutting at the 3' end of the insert (e.g.Notl)
• RNAase inhibitor
• DNAase I, RNAase free (Pharmacia)
Kits • SuperScript plasmid system, pSPORT-1 vector (Gibco/BRL)
Procedure
Toads, oocytes and injections
1. For removal of oocytes, a Xenopus toad is anaesthetized for 15-30 min in
half a liter of ethyl m-aminobenzoate solution.
2. The toad is then placed on top of crushed ice covered with wet tissue
paper, to avoid the skin from drying out. A small incision is made in
the upper skin of about 1 cm length, and a 0.5 cm cut in the inner
skin and the muscular layer.
3. Using forceps, small clumps of oocytes (depending on the amount required, usually 500-1500 oocytes) are removed and are treated twice
for 90 minutes in collagenase A (or D), having low trypsin activity in
a calcium free ORII solution, in order to remove follicular layers. Typically, 10 ml of collagenase solution is placed in a sterile 15 ml tube, and
after cutting the ovarian lobes into small clusters of oocytes, up to 3 ml of
oocytes are added per tube.
4. The tube is rotated in the dark at 18°C on an orbital rotor spinning at low
speed (20 rpm) for 90 min. and then replaced by a new collagenase-ORII
solution for another 90 min.
5. The oocytes are then washed thoroughly 5 times with ORII solution, followed by 5 times with modified Barth's solution.
DANIEL MARKOVICH, ANDREAS WERNER AND HEINI MURER
RN Aaseinhibitor (RNA guard Pharmacia); 50 units T3 or T7 RNA polymerase (Promega).
• phenol:chloroform:isoamylalcohol (25:24: 1)
• collagenase A ( or D ), low trypsin activity (Boehringer, Mannheim; 2 mg/
ml)
• mRNA (of choice)
• Restrietion enzyme cutting at the 3' end of the insert (e.g.Notl)
• RNAase inhibitor
• DNAase I, RNAase free (Pharmacia)
Kits • SuperScript plasmid system, pSPORT-1 vector (Gibco/BRL)
Procedure
Toads, oocytes and injections
1. For removal of oocytes, a Xenopus toad is anaesthetized for 15-30 min in
half a liter of ethyl m-aminobenzoate solution.
2. The toad is then placed on top of crushed ice covered with wet tissue
paper, to avoid the skin from drying out. A small incision is made in
the upper skin of about 1 cm length, and a 0.5 cm cut in the inner
skin and the muscular layer.
3. Using forceps, small clumps of oocytes (depending on the amount required, usually 500-1500 oocytes) are removed and are treated twice
for 90 minutes in collagenase A (or D), having low trypsin activity in
a calcium free ORII solution, in order to remove follicular layers. Typically, 10 ml of collagenase solution is placed in a sterile 15 ml tube, and
after cutting the ovarian lobes into small clusters of oocytes, up to 3 ml of
oocytes are added per tube.
4. The tube is rotated in the dark at 18°C on an orbital rotor spinning at low
speed (20 rpm) for 90 min. and then replaced by a new collagenase-ORII
solution for another 90 min.
5. The oocytes are then washed thoroughly 5 times with ORII solution, followed by 5 times with modified Barth's solution.