Microinjection procedure

Presently, microinjection provides the fastest and simplest means for germ-line transgenesis and transient expression studies in fish (19-22). For more detail concerning microinjection procedures, see Chapter 35 of this volume. 

Definition of the background activity of microinjected oocytes is a clear prerequisite for any functional cloning procedure, as a positive signal needs to be distinguished from the endogenous transport activities. Key characteristics of the carrier, such as a dependency on sodium ions or a specific inhibitor are very helpful. In addition, unspecific binding of the radioactive transporter substrate to the oocyte membrane needs to be examined carefully. 

The first method described was the direct microinjection of the vector DNA with only minor alterations in the target gene into ES cells (Zimmer and Gruss, 1989). Because of the high transfection frequency observed with this method (20%), it was not necessary to use a selection marker. However, this method is technically very difficult to perform and no laboratory could successfully reproduce this procedure, nor have these cells been reported to be transmitted through the germ-line. 

The preferred method for delivery of lentiviral vectors is microinjection into the perivitelline space of single cell or cleavage-stage embryos, which is a far less invasive procedure... 

Need for micromanipulatory procedure. The metabolic studies require the observation of dynamic changes triggered by microinjection of metabolites. The usefulness of both spectral and topographic operation is critically dependent upon the ability to micromanipulate (microinject) living cells, in order to measure changes in injected cells and their neighbors. 

Earlier techniques for measuring cytosolic free Ca2+ (1-2) such as the luminescent photoprotein aequorin, the absorbance dye arsenazo III and Ca2+-sensitive microelectrodes, all required microinjection or impalements, and were therefore applied mainly to giant cells. Later, photoproteins have been loaded with various reversible permeabilization procedures (3), but the largest expansion in the range of cell types in which Ca2+ signals can be quantified has come from the development of new fluorescent indicators that can be loaded using hydrolyzable esters. Currently four fluorescent indicators are used frequently quin-2, fura-2, indo-1 and fluo-3. 

The ES cell aggregation method, on the other hand, is a relatively easy technique, and does not need expensive equipment or extensive experience, but demands higher quality ES cells (early passage cells are required). This method was developed by Nagy et al. (27). After microinjection or aggregation, blastocysts are transferred into the uterus of pseudopregnant mice using standard procedures (26). 

The majority of the transgenic mice produced to date have been through the microinjection route, which was used for the production of the first transgenic mouse (Isola and Gordon, 1991). In this procedure, fine glass micropipettes are used to introduce extremely small volumes of DNA solutions into the pronucleus of... 

Plant transformation techniques allow the delivery of the transforming DNA through the cell wall and plasma and nuclear membranes, without compromising the viability of the cell. Gene delivery can be performed either via a biological vector (plant viruses or bacteria) or by non-biological vector-free procedures (chemical methods, microinjection, particle bombardment, etc.) (Birch 1997). 

Currently, it is standard procedure to develop ion channel-specific antibodies for immunocytochemistry, to perform Western and Northern blot analyses, ion channel in situ hybridization, or reverse transcription polymerase chain reaction (RT-PCR). The introduction of the single-cell RT-PCR in combination with the patch-clamp method in the 1990s made it possible to identify gene transcripts and to correlate them with functional data for the same individual cell. Finally, one of the most powerful cell biological techniques in the study of ion channels is based on artificial expression systems such as microinjection of mRNA encoding channel subunits into Xenopus oocytes and selective expression of native ion channels or with different subunit composition (e.g., Ky channel subunits). Because the Xenopus oocytes are large, they are a perfect model to study artificially expressed channels. Another good model for artificial ion channel expression is the Chinese hamster ovary (CHO) cell line. 

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