USE OF CHAPOL
CHAPOL was used to infect the developing brain of chick embryos using the procedures outlined above. At various times later, the tissue was harvested and stained for AP activity. The outline of each section was drawn by camera lucida and the location and type of cells labeled on each section were recorded. A single cell or cluster of cells with a small group of surrounding cells were removed using a heat pulled glass micropipette (figure 2) and transferred to a 96-well PCR plate for Proteinase K digestion (as below). Following digestion, nested PCR was performed (as below). The product of each PCR was run on a 1.5% agarose gel to determine if a product of the appropriate size had been amplified. The recovery of a PCR product of the proper size occurred from PCR of 30-85% of the picks (the frequency varied depending upon the batch of PCRs and the tissue being studied) using CHAPOL. Sequencing of the oligonucleotide insert (as below) was performed on all reactions which gave the expected product on the agarose gel analysis (e.g. figure 3) and was successful approximately 75% of the time. All sequences were stored in the software program GCG (1991). All common sequences were pulled from the database created in GCG and the corresponding cells labeled. Sections were then aligned to determine the three dimensional boundaries of clonal expansion. Each type of cell (e.g. neuron, glia) was also recorded to determine the variety of cells which can arise from a single progenitor.
Figure 2:Picking of AP+ cells from tissue sections following infection with CHAPOL. (A) Several AP+ cells are present in a chick cerebellar section, including a Purkinje cell (arrow) and many glial cells. (B) After removal of the Purkinje cell using a glass micropipette.
Figure 3:The sequence reactions from the PCR products of four representative samples (samples 1-4) are shown. Sequencing of PCR products 1 and 2 each revealed the presence of a unique sequnce. Sequencing of PCR products 2 and 4 yielded the same sequence. Sequencing of sample 3 showed the presence of more than one species.
The value of using a complex library of vectors is illustrated by the view of more heavily infected brains (figure 4) where closely aggregated AP+ cells would have been lumped into a single clone based on proximity. In addition, even lightly infected brains could give rise to lumping errors (also see Walsh and Cepko, 1992 [41]).
Figure 4:A 60 mm parasagittal section of a chick cerebellum from a brain infected with CHAPOL and analyzed at P14. The AP+ cells were removed, subjected to PCR, and the PCR products were sequenced. Each pick that yielded a PCR product is labeled by an arrow. The sequence identity of each PCR product is color-coded. The clone indicated by the magenta arrows were not closely clustered, and would have resulted in a splitting error if certain geometric critera were used in clonal definition. The clones indicated by the blue and the green arrows were located very close to each other, and would have resulted in a lumping error if certain geometric criteria of clonal assignment were used.
Two issues are important for determining the value of this type of library of DNA markers: the number of unique members in the library and the distribution of the library members [42]. If only 2 members exist in the library, for example, then there is a one in two chance that the same tag will be selected in two consecutive picks (if they are present in equal concentrations). If 100 members exist in the library at equal concentrations, the chance that two picks come up with the same member is reduced to 10-2. The second important variable determining the quality of the library is the distribution of the members within the library. This can be illustrated as follows. Consider a library composed of 106 members, with 50% of the library composed of one member. If two neighboring or distant cells are found to carry the over represented insert, the probability that the two cells arose from separate clones is still 0.5. CHAPOL was found to have an equal distribution in that each of the inserts picked to date (n> 500) has occurred independently only once. One further issue to consider is the level of difficulty in using the library. We have found in practice that this method of tag identification is in fact easier than our previous method based upon the analysis of the size and restriction digestion pattern of A. thalliana DNA.
