3. At approximately 18 to 42 hours incubation (Hamburger and Hamilton stage 10-17 [38]) embryos are injected with 0.1 to 1.0
Infected chick or rodent embryos can be incubated to any desired point. Chicks can be allowed to hatch and rodents can be delivered by Caesarean section and reared to maturity. Embryonic brains are dissected in PBS followed by overnight fixation in 4% paraformaldehyde in PBS (pH 7.4) at 4°C. Posthatch or postnatal animals are perfused with the same fixative and are incubated overnight in the fixative. They are then washed overnight in three changes of PBS and cryoprotected in 30% sucrose. After cryoprotection, the brains are embedded in OCT media and cut on a Reichart-Jung 3000 cryostat at 60-90
mm. Sections are histochemically stained for the appropriate marker and are mounted with gelvatol. For details of the histochemical reaction for lacZ or PLAP, see Cepko et al. in [40].Solutions
PBS (lOX):Glutaraldehyde Fixative (0.5%)
Paraformaldehyde Fixative (4%):
Heat H20 to 60°C. Add paraformaldehyde. Add NaOH to get paraformaldehyde in solution. Cool to room temperature, add 1/10 volume 10X PBS and pH with HCl. Can be stored at 4°C several weeks.
PREPARATION AND USE OF A RETROVIRAL LIBRARY FOR LINEAGE ANALYSIS USING PCR AND SEQUENCING
We developed a direct approach to address lumping and splitting errors [41] by constructing a library of viruses that was analyzed by PCR. In our first libraries, each virus of the library carried one member from a pool of approximately 100 DNA fragments from Arabidopsis thalliana DNA, in addition to the lacZ or PLAP gene. Infected cells, recognized by their enzyme activity, were mapped and the positive cells cut from cryosections. The A. thalliana DNA was amplified by PCR and characterized by size and restriction enzyme digestion patterns. If the size and restriction digestion pattern of the PCR product from two or more cells was the same, they were considered siblings with a probability calculated on the basis of the number of infections in that brain and the complexity of the library [41, 42]. Lineage analysis using such libraries revealed novel lineal relationships in the rat cerebral cortex [41, 43, 44] and chick diencephalon [45]. However, the limited number of unique members in the library made from A. thalliana DNA restrained the analysis to tissues with low infection rates.
More data could be acquired with each experiment and additional questions could be addressed in the central nervous system and other tissues with a more complex library containing a greater number of DNA tags. We therefore constructed several retroviral vectors, of which CHAPOL (chick alkaline phosphatase with oligonucleotide library) is the prototype [46], that include degenerate oligonucleotides with a theoretical complexity of 1.7 x 107. Studies in the developing nervous system of the chick have been successfully completed using CHAPOL [47, 48].
A summary of the production of CHAPOL and BOLAP (an oligo library in a murine vector) will be given here; a detailed description of the construction of CHAPOL can be found elsewhere [46]. For either avian or murine retroviruses, the overall strategy is the same. A population of double-stranded DNA molecules that includes a short degenerate region, [(G or C)(A or T)]12, is generated by PCR amplification of a chemically constructed single-stranded oligonucleotide population of the same sequence. The oligo preparation is ligated into a retrovirus vector and a preparation of highly competent E. coli is transformed. The library is then grown as a pool and a preparation of plasmids from the pool is made. The DNA of the pool is transfected into an avian or mammalian packaging cell line to produce a library of virus particles. The library is injected into an area to be mapped. Infected cells are detected histochemically and each infected cell is recovered for PCR amplification. Each PCR product is then sequenced. Two cells with the same sequence are considered siblings, again with a probability derived from an analysis of the frequency of recovery of each genome (see Walsh et al. 1992 [42]).
PREPARATION OF CHAPOL
The avian replication-incompetent virus CHAP [49, 50], encoding the human placental alkaline phosphatase (PLAP) gene, was modified to accept the oligo inserts. CHAP was linearized, purified, and mixed with the degenerate oligonucleotides in the presence of ligase and aliquots of the resulting ligation products were used to transform E. coli DH5
a. Following transformation, all aliquots were pooled. One hundred ml of the pool was plated at varying dilutions on plates containing ampicillin. The remainder of the pool was divided and added to eight 2L flasks containing lL LB media with 50 mg/ml ampicillin. The cultures were shaken overnight at 37°C. Plasmid DNA was extracted from these cultures by the triton lysis procedure and purified on CsCl gradients [51].CHAPOL DNA was transfected into the avian virus packaging line Q2bn [14], and the transiently produced virus was collected and concentrated. Aliquots of CaPO4 precipitates of 100
mg CHAPOL DNA were made in 10 ml of HBS. The precipitate in each aliquot was then distributed equally on ten ten cm plates of Q2bn and glycerol shock was carried out for 90 seconds at room temperature 4 hours later. At 24 hours post glycerol shock, the supernatants were collected and pooled. This was repeated at 48 hours. The supernatants from the 24 and 48 hour harvests were pooled and the titer calculated by infection of QT6 cells and assay of the PLAP activity as described (Cepko and Pear in Ausubel et al. 1997 [4]). The stock was filtered through a 0.45 mm filter and concentrated by centrifugation in an SW27 rotor at 4°C, 20K, for 2 hours. The concentrated stock was titered on QT6 and tested for helper virus, which proved negative. The titer of CHAPOL was determined to be 1.1 x 107 CFU/ml. The same stock has used for all experiments conducted over a 5 year period and many aliquots remain. We recommend making large stocks and storing them as small aliquots.
