Embryo injection procedures

For microinjection experiments in fish, a glass needle loaded with DNA solution is used to penetrate the plasma membrane of a one-cell-stage embryo. The injection procedure is performed with the aid of a micromanipulator under a standard dissecting microscope. Once the tip of the needle has entered the cytoplasm, approximately 1-2 nL of DNA solution containing 10 to 10 DNA molecules is injected. 

Enhancer-dependent expression is observed following transplantation of an injected pronucleus into a two-cell blastomere (Henery et al., 1995) (Figure 4). The first mitosis—and concomitant loss of the plasmid from the injected pronucleus—was circumvented by the transplantation procedure and hence cannot account for the enhancer requirement observed in the two-cell embryo. The luciferase reporter gene is driven by the / promoter, which has two Spl binding sites (termed the proximal and distal sites)—a CAAT box binding site that binds CTF and a TATA box that binds TBP. Analysis of the expression of a set of linker scanning mutations that inactivated each of these sites without altering the distances between each of the promoter elements revealed that each mutation had the same relative effect on tk... 

The procedure for making injections of retrovirus into the telencephalic ventricles of embryonic mice and rats will be described. The basic procedures can be adapted to inject retrovirus into other structures, such as the eye (15) or olfactory epithelium (16,17). Intraventricular injections of retrovirus can be made most readily between embryonic d 11 and 15 of mice and between embryonic d 13 and 17 of rats. Injections earlier and later than the stated times are difficult because the uterine membranes are somewhat opaque, making it difficult to resolve the enclosed embryo. Furthermore, at earlier times the embryo, as well as its brain is very small and difficult to aim for with the tip of a micropipet (see Note 28). Nevertheless, the particular question under investigation will determine the precise developmental age at which the rodent embryo is injected with recombinant retroviruses. 

Fig. 2. In-ovo electroporation. A pair of electrodes held by a manipulator (A) is inserted intom a window opened on the shell (B). The electrode is placed on the vitelline membrane overlying the embryo (C), and a 25-V 50-ms pulse is charged five times. The entire procedure is monitored under a dissection microscope. Plasmid solution is injected to the E2 (HH stage 10) chick neural tube (D) prior to the pulse charge.The dimensions of the electrode are shown in E. Most of the electrode is insulated (black in the figure) so that only the tip is exposed (white area). One hour after electroporation, some embryos were fixed, processed for paraffin sectioning, and observed with a Nomarski interference microscope (F). The right-hand side of the figure corresponds to the right of the embryo, where injected plasmid was transfected. The morphology of the cells and the structure of neural tube were almost normal. The blue deposit inside the neural tube is a complex of plasmid and the color substrates not removed by washing in dimethylformamide after whole-mount in-situ hybridization. Twenty-four hours after electroporation, the development of yolk sac plexus, vitelline veins, and vitelline arteries are retarded in the area contacted on the electrodes (arrows in G). Bar is 2mm (C) 50 pm (F) 4mm (G). Source (3).

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