In 1974, Rudolf Jaenisch created the first mouse carrying a foreign gene by injecting the DNA of the SV40 virus into the blastocyst of the mouse. Later, researchers injected Murine leukemia virus into mouse embryos to obtain mice that can be stably inherited through the reproductive system, and the foreign genes can be stably expressed in the offspring. These mice that are stable in inheritance and express foreign genes are the transgenic mice that we now generally call.
Method principle
The basic substance of heredity is DNA. A gene is a DNA fragment that has a genetic effect on a chromosome. It can be called a genome for all genetic information stored in a complete set of chromosomes. The genetic composition of different species and different individuals is different. For an individual animal, the non-self genetic component belongs to a foreign gene. If the foreign gene is integrated or introduced into the animal chromosome gene, the foreign gene is called Transgene (the transferred gene), this animal is a transgenic animal.
Material
Mouse, HCG, PMSG, hyaluronidase sodium, pentobarbital, microscope, scorpion, egg holding needle, injection needle
Steps
1. 7-8 weeks old female mice were selected and the vaginal opening was closed. As a donor, PMSG (10 IU) was injected intraperitoneally into each mouse at around 3:00 pm.
2. After 47~48 hours, each mouse was intraperitoneally injected with HCG (0.8 IU) and caged with normal male rats. Several female rats of appropriate age (above 2 months) were used as recipients, and the vaginal opening was flushed. Ligation of the male mouse cage.
3. Observe the donor and recipient before 9:00 am the next day. The receptor cage is taken out as a measure of isolation.
4. At about 10:30, the donor was sacrificed by cervical dislocation. The entire fallopian tube was removed by surgery and placed in hyaluronan~0.3 mg/M2 solution. Under the microscope, the ampulla of the fallopian tube was torn open with tweezers, and the fertilized eggs flowed out along with the granulosa cells.
Precautions
1. The needle tip should be kept away from personnel and instruments in the laboratory throughout the process. When the needle is not securely attached to the needle holder, the pressure in the syringe, tube and needle may cause the needle to be ejected.
2. The injection rate is too fast to disturb the cytoplasmic components, cell lysis or cell migration from the bottom layer. The injection effect is best when the injection volume is less than 50% of the estimated cell volume and the flow rate of the injected sample hardly results in a visible cytoplasmic shift.
3. Ultraviolet light can be used to see the living cells after the injection of the marker, but it will cause irreparable damage to the cells, so the UV irradiation time should be shortened as much as possible.
Other tips
1. The optimal composition varies significantly depending on the particular cell type used. If efforts to optimize voltage and pulse width electroporation results are still unsatisfactory, try changing the perforated media.
2. Another important factor affecting electroporation/electrofusion is related to cell state. To achieve maximum efficiency, cells in the middle of logarithmic growth must be collected.
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