The term “transgenic animals” describes animals whose chromosomes contain stably integrated copies of genes or gene constructs derived from other species or not normally found in the host animal.
For this reason, mice, rats or other small mammals are used to introduce foreign DNA into oocytes or embryos (blastocysts).
Two different techniques are used:
Microinjection of linear DNA into the pronucleus
Microinjection of DNA
Injection of linear DNA molecules into fertilized eggs (pronuclear stage) using an inverted microscope, micromanipulation equipment and injection / holding devices.
The first successful production of transgenic mice using pronuclear microinjection was reported in 1980 [1]. The pronuclear microinjection method of producing a transgenic animal results in the introduction of linear DNA sequences into the chromosomes of the fertilized eggs. If this transferred genetic material is integrated into one of the embryonic chromosomes, the animal will be born with a copy of this new information in every cell. The foreign DNA must be integrated into the genome prior to the doubling of the genetic material that precedes the first cleavage.
If this does not occur, only a few cells will integrate the gene. For this reason, the DNA is introduced into the fertilized egg at the earliest stage, which is the pronuclear period immediately following fertilization. For several hours following the entry of the sperm into the oocyte, the male and the female pronuclei are visible as individuals under normal light microscope and not fused into a so-called zygote.
The DNA may be injected into either of these pronuclei with no difference in results. Usually the injection is into the male pronucleus because it is larger than the female nucleus and also because it is closer to the oocyte surface.
These oocytes are subsequently transferred into the uterus of pseudopregnant recipient animals and develop to term.
Setup of the workstation:
One TransferMan micromanipulator is used for moving the holding capillary, the other TransferMan is used for moving the injection capillary.
The actual holding of the embryo is carried out using CellTram Air.
Two possibilities are available for injecting DNA: using the CellTram Air as a simple injection device or using the FemtoJet™ for reproducible and programmable injections.
Together with an inverted microscope, combined with Hoffmann modulation contrast optics or differential interference contrast optics, microinjection can be effected quickly and simply.
Preparation of the object slide:
One drop of medium is placed on the slide. The embryos to be injected (10 to 20) are placed in one area. The whole droplet is covered with oil.
Operating:
Operating TransferMan, by means of four self-explanatory keys, is extremely simple. For every TransferMan device, only two spatial coordinates are defined and stored.
The capillary can be moved easily in any direction (x y z) by means of a joystick. By simply pressing the joystick button, the capillary can be moved repeatedly to one of the two pre-programmed positions.
The setting of these positions depends on how the TransferMan is used (Fig. 4)
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