The technique of introducing new genetic material into the germline of mammals has been a major development in biotechnology over the last decades.
“Transgenic animals” are animals whose chromosomes contain stable, integrated copies of exogenous genes, additional copies of endogenous genes or gene constructs.
They are frequently created by two different techniques:
1) microinjection of DNA into the pronucleus of zygotes and 2) injection of embryonic stem cells into blastocysts.
In this Application Note, these techniques, as well as another technique for creating chimeras, the production of tetraploid mouse embryos, are discussed.
The characterization of molecular genetic changes of specific cell populations is important for the understanding of a developing tumor. A rapid one-step microdissection technique applied for the isolation of tissue areas has been developed.
The use of the oscillating MicroChisel allows precise microdissection of cells for analysis and their easy detachment from the glass slide. The time necessary for preparation will vary depending on the number of cells cut and the tissue architecture.
Preparation with the oscillating MicroChisel allows a sharp separation between the dissected area and unwanted tissue that remains intact for further analysis.
With the help of our ECET device the Eppendorf MicroDissector, J. Rohwedel and C. Kruse, isolated dense cell areas from living tissue derived from embryonic stem cells (mouse) and could successfully show their individual cultivation.
Beside the experimental description, the application contains a userguide, explaining the microdissection procedure step by step.
With the help of the Eppendorf MicroDissector, which
uses the so called Piezo-Power Microdissection (PPMD),
the isolation of dense cell areas from living tissue for further
individual cultivation has been successfully shown by
Rohwedel and Kruse.
In this application note is described how the microinjection procedure into hydra embryos is performed with the aid of an Eppendorf Micromanipulator 5171, CellTram vario and a Zeiss Axiovert 100 inverted microscope.
This technique can be employed for assisted conception in animals in where standard ICSI fails, such as mice. The microinjection workstation required for this technique is similar to standard ICSI, but with the addition of a piezo impact unit attached to the universal holder. The device vibrates the injecting micropipette axially and drills its way into the oocyte. This method has been shown to increase success rates.
Recently an alternative procedure to Tetraploids to generate mice entirely derived from injected ES cells was described (Gridley, T. & Woychik, R. (2007), Poueymirou, W.T. et al. (2007)). The ES cells are injected into embryos at the eight-cell stage as this is suggested to result in a higher degree of ES cell contribution.
Microinjection techniques are widely applied in developmental biology for the analysis of early developmental processes such as gastrulation, neural induction and patterning or organogenesis. Microinjection experiments into vertebrate embryos (e.g. mouse, frog, fish) allow to generate transgenic animals by injection of DNA, to interfere with specific developmental processes (DNA, RNA, morpholino-injection), or to follow the fate of individual cells by the injection of fluorescent lineage tracer dyes.