METHODS FOR DETERMINING LINEAGE ANALYSIS
In order to determine the ratio of 2 genomes present in a mixed virus stock (e.g. BAG plus DAP), there are several methods that can be used. The first 2 methods are performed in vitro, and are simply an extension of a titration assay. Any virus stock is normally titered on NIH 3T3 cells to determine the amount of virus to inject. The infected NIH 3T3 cells are then either selected for the expression of a selectable marker when the virus encodes such a gene (e.g. neo in BAG and DAP), or are stained directly, histochemically, for
b-gal or PLAP activity without prior selection in drugs. If no selection is used, the relative ratio of the two markers can be scored directly by evaluating the number of clones of each color on a dish. Alternatively, selected G418-resistant colonies can be stained histochemically for both enzyme activities and the relative ratio of blue vs. purple G418 resistant colonies computed. A third method of evaluating the ratio of the 2 genomes is to use the values observed from in vivo infections. After animals are infected and processed for both histochemical stains, the ratio of the 2 genomes can be compared by counting the number of clones, or infected cells, of each color. When all the above methods were applied to lineage analysis in mouse retina [20] and rat striatum [34], the value obtained for the ratio of G418 resistant colonies scored histochemically was almost identical to the ratio observed in vivo. Directly scoring histochemically stained, non-G418 selected NIH 3T3 cells led to an underestimate of the number of BAG infected colonies, presumably as such cells often are only faint blue, while DAP-infected cells are usually an intense purple. In vivo, this is not generally the case as BAG-infected cells are usually deep blue.The method of injecting 2 distinctive viruses is a straightforward and feasible method of assessing clonal boundaries when they are fairly easy to define. This method does not require circumstances where there is a wide range of dilutions that can be injected to give countable numbers of events, which is required for a reliable dilution analysis. Moreover, it does not rely as critically on controlling the exact volume of injection, as is required in the dilution analysis. The use of a small number of vectors that encode distinctive histochemical products for definition of sibling relationships is only appropriate when there is very little migration of sibling cells. In these cases, the arrangement of cells that will be used to identify clonal relationships can be defined, and then this definition can be tested as described above. The fit of the definition with true clonal relationships will be revealed by the percentage of defined "clones" that are of more than one color. When the error is too great, one can re-evaluate the criteria, make a new definition, and again test it by looking for "clones" that are more than one color. Through trial and error, an accurate definition of sibling relations should be possible when migration is not too great.
When one cannot accurately define clonal relationships with a few distinctive viruses, a much greater number of vectors must be used. One can employ a library of retroviral vectors, each member of which is tagged with a unique small insert of an irrelevant DNA. Each vector is scored using the polymerase chain reaction (PCR). The library/PCR method is tedious but extremely worthwhile when dealing with problematic areas.
Regardless of which method is used to score sibling relationships, one further recommendation to aid in the assignments is to choose an injection site which will allow the inoculum to spread. If one injects into a packed tissue, the viral inoculum will most likely infect cells within the injection tract and it will be very difficult to sort out sibling relationships. For example, a lumen, such as the neural tube, provides an ideal site for injection. Regardless of site, one must inject such that the virus has clear access to the target population; the virus will bind to cells at the injection site and will not gain access to cells that are not directly adjoining that site.
The procedures described below are those that we have used for infection of rodents and chick embryos, histochemical processing of tissue for
b-gal and PLAP, and preparation and use of a library for the PCR method of defining clonal relationships.INFECTION OF RODENTS
Injection of virus in utero in rodents
The following protocols may be used with rats or mice. Note that clean, but not aseptic, technique is used throughout. We routinely soak instruments in 70% EtOH before operations, use the sterile materials noted, and include Penicillin/Streptomycin (final concentrations of 100 units/ml each) in the lavage solution. We have not had difficulty with infection using these techniques.
Materials
1. Mix ketamine and xylazine 1:1 in a 1 ml syringe with 27-gauge needle; lift the animal's tail and hindquarters with one hand and with the other inject 0.05 ml (mice) or 0.18 ml (rats) of anesthetic mixture intraperitoneally .
One or more additional doses of ketamine alone (0.05 ml for mice and 0.10 ml for rats) is usually required to induce or maintain anesthesia, particularly if the procedure takes over one hour. Respiratory arrest and spontaneous abortion appear to occur more often if a larger dose of the mixture is given initially, or any additional doses of xylazine are given.
2. Remove hair over entire abdomen using depilatory agent (any commercially available formulation, such as Nair, works well); shaving of remaining hair with a razor may be necessary. Wash skin several times with water, then with 70% EtOH, and allow to dry.
3. Place animal on its back in support apparatus.
For this purpose, we find that a slab of styrofoam with two additional slabs glued on top to create a trough works well for this purpose. With the trough appropriately narrow, no additional restraint is then needed to hold the anesthetized animal.
