Trouble Shooting

Since many people have run into difficulty getting the transgenic procedure working well in their labs, please find below a few general guidelines which should improve efficiency. If the information on this page does not answer your particular questions, please feel free to email me.

You should be able to get a high frequency of normal embryos that are transgenic: in my lab we routinely generate 500-700 embryos from nuclear transplantations per day, most which developed normally to advanced stages (feeding tadpoles). I believe the main reason for poor results is that the nuclei becomes damaged during the procedure and therefore the resulting embryos are severely aneuploid. The most common result from damaged nuclei are embryos that arrest during the gastrula stages.

Decondensed sperm nuclei are VERY fragile. Therefore there are several things to consider once the sperm nuclei are added to the egg extracts. For example, decondensed sperm nuclei can be damaged by mixing, being placed on ice, front filling, or using needles that are too small. Also with time, decondensed nuclei are less and less likely to give normal development. Therefore, follow these general rules to keep sperm nuclei relatively intact:

• At all steps, mix sperm suspension by gently pipetting up and down with a yellow tip that has been clipped at the end
• Keep decondensed sperm nuclei at room temperature
• Decondensed sperm nuclei must be transplanted within 1 hour, preferably within 30 minutes
• Back-fill sperm nuclei into needle, rather than front-fill
• Use 80-100 micron inner diameter bevelled needle point

We usually do three to five separate reactions each with a different construct (so that each experiment has controls, etc). This is how we do it:

• First we take out some sperm dilution buffer from the freezer and allow it to equilibrate to room temperature (so that when we dilute the reaction, the nuclei are at room temp).
• Then we mix the condensed nuclei and linearized DNA and incubate 5 min at RT.
• During these five minutes we get one 25ul extract aliquot from the freezer and put it in a rack at RT until it melts.
• Also we make a dilution of the enzyme.
• After the 5 minute timer goes off, we add the diluted enzyme, the Mg2Cl and the extract to the nuclei-DNA mixture. We mix the reaction with a cut-off yellow tip by gently pipetting up and down. Be careful not to mix too hard or make bubbles in the tube (surface tension is deadly to nuclei!).
• We incubate the mixture for 15 minutes at room temperature.
• During this incubation we strip eggs from one or two frogs and dejelly them in 2% cysteine (in 1XMMR pH7.8-8.0). This usually takes about 10 minutes, so by the time the eggs are ready, the reaction is nearly done.
• We dilute the reaction, mix gently with a cut yellow tip and back load a needle for transplantation.

We usually transplant for about 30 minutes. During this time one person can transplant several hundred nuclei. Then we start the next reaction. Go through that reaction like the one before and then do the next one, etc. With a little practice and good organisation, you can get it to move quite smoothly from one reaction to the next. It usually takes slightly less than an hour between one reaction and the next. It works better if two people work together, not only because it is less hectic, but also because you can get twice as many transplantations done. Organising the experiment one or two people can transplant a thousand or more nuclei and at no time have a nucleus spend more than 30 minutes between decondensation and transplantation. Also the hour between each transplantation gives the frogs plenty of time to make more eggs. Finally by the time the last transplantations are done, the first batch of transplanations are old enough to be screened. It is a full day's work so lately we have been making the sperm nuclei the afternoon before and keeping the condensed nuclei in storage buffer in the refrigerator until the next morning. In fact, we usually store the nuclei in the refrigerator for two days so that we can do two full days of transgenic experiments from one sperm nuclei prep.

The injection apparatus we now use is a Harvard Apparatus infusion syringe pump (Model 22). It works great for nuclear transpantations. It can be set at any desirable flowrate. We use 10nl/sec. Ever since we started using the pump we have been getting very high frequencies of normal transgenic embryos. We have the system set up with two 2.5ml Hamilton Gas Tight Syringes and Tygon R3603 tubing (0.8mm X 0.8mm). This way two people can transplant nuclei at the same time. The oil that we use to fill the system is Mineral Oil (Embryo Tested) from Sigma (Cat. No. M-8410).