转基因动物protocol -转基因动物-生物通

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TMF Protocols

For more information about making, genotyping, and breeding transgenic animals, the following references are recommended:

Manipulating the Mouse Embryo, 2nd Ed., by B. Hogan, R. Beddington, F. Costantini, and E. Lacy. Cold Spring Harbor Laboratory Press, 1994.
Transgenic Animal Technology, edited by C. Pinkert. Academic Press, Inc., 1994.

Gene Targeting: A Practical Approach, edited by A. L. Joyner. Oxford University Press, 1995.

Strategies in Transgenic Animal Science, edited by G. M. Monastersky and J. M. Robl. ASM Press, 1995.

Mouse Genetics: Concepts and Applications, by Lee M. Silver. Oxford University Press, 1995.

Mouse Genetics and Transgenics: A Practical Approach, edited by I. J. Jackson and C. M. Abbott. Oxford University Press, 2000.

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PREPARATION OF DNA FOR MICROINJECTION
One of the most critical steps in making transgenic mice is preparing the DNA for microinjection. Poorly prepared DNA can be toxic to the ovum and contaminants can clog the injection needle, which typically have inside diameters of 0.5 micron at the tip. Therefore, the TMF suggests purification of DNA for pronuclear injection by one of the following protocols.
Protocol 1 (Organic Extraction)

Use a restriction enzyme that removes as much of the vector sequence as possible, because plasmid DNA is toxic to the ovum. Cut the DNA and electrophorese through low-melt agarose (we recommend Seaplaque) in 1X TAE (Maniatis) without EtBr, at 30-40 mA. Load no more than 500 ng per lane. Stain the gel in EtBr (1 μg/ml) in the dark and visualize the bands with long wavelength UV, keeping exposure of the gel to UV light at a minimum to avoid nicking the DNA. Cut out the insert band and melt the agarose. Extract twice with phenol (prepared per Maniatis), adding 200 μl TE after the first extraction. Extract once with chloroform. Precipitate with isopropanol, wash once with 70% EtOH, and dissolve the pellet in 25 μl water. Analyze on a 0.8% agarose gel to check the purity of the DNA. If you run several volumes of DNA (e.g., 1-3 μl) with serial dilutions of a standard of known concentration, you can also determine the concentration from this gel. A picture of this gel and the concentration of the DNA must be supplied to the TMF. The final dilution of the DNA will be done by the TMF.

Protocol 2 (no phenol)

Use a restriction enzyme that removes as much of the vector sequence as possible, because plasmid DNA is toxic to the ovum. Cut the DNA and electrophorese through low-melt agarose (we recommend Seaplaque) in 1X TAE (Maniatis) without EtBr, at 30-40 mA. Load no more than 500 ng per lane. Stain the gel in EtBr (1 μg/ml) in the dark and visualize the bands with long wavelength UV, keeping exposure of the gel to UV light at a minimum to avoid nicking the DNA. Cut out the insert band and purify using the Bio101 GeneClean Spin kit. Load about 200-300μg of the gel on each spin column--usually a fragment will require 4-6 columns to take care of all the gel. Add glassmilk and melt at 55C for 5 min, flicking the tube at least every 1-2 minutes. Spin out the solution for 30 sec, wash 2X with NEW wash, spinning 30 sec each time, dry the filter by spinning for 1 min, and then elute the fragment with 10-20 μl of elution buffer, incubating at 37C for 5 min (flick the tube at least 2X in that period), spinning down, and then repeating to get any remaining DNA. Precipitate with isopropanol and wash twice with 70% EtOH. Dissolve the pellet in 25 μl water. Analyze on a 0.8% agarose gel to check the purity of the DNA. If you run several volumes of DNA (e.g., 1-3 μl) with serial dilutions of a standard of known concentration, you can also determine the concentration from this gel. A picture of this gel and the concentration of the DNA must be supplied to the TMF. The final dilution of the DNA will be done by the TMF.

Protocol 3 (Qiagen kit)

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PREPARATION OF DNA FROM TAIL CLIPPINGS
Anesthetize the mouse, identify it by ear punching, and remove 1-1.5 cm of tail with a sterile razor blade. The end can be cauterized by heating the blade, but this is not usually necessary. Place the tail piece in a 1.5 ml microfuge tube and add 0.5 ml of 50 mM Tris (pH 8), 100 mM EDTA, and 0.5% SDS. Add 25 μl of a 10 mg/ml solution of Proteinase K and mix well. Incubate at 55C overnight. If the tail is not totally submerged, the tube should be on a rocking platform. Add 0.5 ml phenol (equilibrated with Tris, pH 8), and shake vigorously for 3 minutes. Centrifuge at high speed for 3 min to separate phases. Transfer the aqueous phase to a clean tube, avoiding the interface. Add 0.5 ml phenol/chloroform (1:1) to the aqueous phase and mix vigorously for 2 min. Centrifuge 2 min and transfer aqueous phase as before. Add 50 μl of 3M sodium acetate (pH 6) and 0.5 ml of 100% ethanol (room temp). Invert to mix thoroughly. The DNA should immediately form a visible precipitate. (If a pH below 6 is used, the EDTA will also precipitate.) Pellet the DNA for 30 sec and decant or aspirate the ethanol. Add 1 ml of 70% ethanol and vortex. Centrifuge for 1 min, remove the ethanol, and dry the DNA under vacuum for 4-5 min. Redissolve in 100 μl TE (10 mM Tris, pH 8, and 1 mM EDTA). This may require overnight incubation, or heating to 65C for 5-10 min. Depending on yield, 10-20 μl should be sufficient for a Southern blot, and 1-2 μl for PCR.

Dos and Don'ts of Mouse Breeding
Do minimize the amount of noise and traffic in your mouse room. Try to always have the same person(s) take care of your mice. Ultrasonic noise, in particular, can disrupt normal mouse behavior (including mating behavior), and can be produced by something as innocuous as a dripping faucet. Contact the TMF about borrowing an ultrasound detector to test your mouse environment.

Do get to know your husbandry staff and maintain a good working relationship with them. They will usually be the first people to notice when something is wrong with your mice.

Do make use of the box at the bottom of the standard cage card to identify the mice in that cage. The information in this box will appear on any Animal Health Reports filed by ULAR (University Lab Animal Resources) staff.

Don't bang cages around or jostle the mice any more than necessary. Handle them calmly and quietly.

Do allow male mice a little time (e.g., a day) to get used to a new cage before expecting them to mate successfully. Males like to mark their territory before "getting down to business".

Don't let mice get too old prior to first mating, if you are planning to breed them. It's best to give them at least some practise within 4 months of birth.

Don't expect breeding females to be productive after more than 8 months of age, although they can have litters well beyond that. Males are generally productive at least a year, and often well beyond that. Maximize production of pups by replacing your breeders with younger mice on a regular schedule.

Do keep records of breeding performance so time and resources aren't wasted on unproductive mice. Not all mice will breed successfully, even among standard strains.

Do cycle females through a male's cage to maximize production from a small number of males. The estrus cycle is 4-5 days long, so females should be left in the cage at least this long, unless you are checking for plugs.

Don't cycle males through a female's cage - pheromones from new males can disrupt pregnancies initiated by previous males.

Don't disturb gestating females any more than necessary, especially a few days before and after birth.

Do use "Do Not Change" cards on the cages of gestating females to minimize disturbances a few days before and after birth. If a cage needs changing while a Do Not Change card is on it, you will be responsible for changing the cage.

Do leave a female with the same male continuously to take advantage of postpartum estrus. The female can get pregnant within 24 hours of giving birth and normal males will not harm their pups. If you have plenty of males, 1:1 matings are recommended to maximize production while minimizing labor.

Do use dirty bedding from a male's cage to stimulate estrus in females. Females group-housed for long periods can go into anestrus, i.e., they will stop cycling normally. This is reversed by exposure to male pheromones present in the urine.

Do check for vaginal plugs if you want to know exactly when a female has mated successfully. This is best done in the morning because the plugs will start to fall out after a few hours. The presence of a plug is not absolute proof of pregnancy, but it is a very reliable indicator. On the other hand, the absence of a plug is not a guarantee that the female isn't pregnant.

Do communicate with the vivarium manager and husbandry staff about any special precautions for your mice, e.g., autoclaved water, cages, and bedding, and irradiated (or autoclaved) feed for immunocompromised mice. Special diets are available and those that aren't can be custom made by the manufacturer.

Do give your mice some environmental enrichment. Nestlets are an inexpensive choice - they are compressed cotton squares that mice love to tear apart and fluff up into a warm nest. Other options include plastic or cardboard tubes, or "shelters". Nesting material of some sort should be given to all breeding females.

Do keep your mice on a regular light/dark cycle with at least 12 hours of light. The TMF uses a 14-hour light cycle. Periodically check that the lights actually come on and go off as scheduled. Avoid entering mouse rooms during dark periods. A red light can be used to minimize impact on the mice if you have to work in the room during dark hours.

Don't transfer mice from one room to another without permission from a UCI veterinarian. It is ULAR policy to have all animal transfers performed by vivarium staff.