Any of the aforementioned packaging systems and vectors can be used to produce vector stocks for lineage analysis. All stocks should be assayed for the presence of helper virus. A detailed description of protocols for making stocks, for titering and concentrating them, and checking for helper virus contamination, has been published and will not be given here (see Cepko and Pear in Ausubel et al., 1997 [4] and reference [5]). The components for producing such stocks are generally available from the laboratories that have constructed them. For example, we have deposited stable lines that produce the murine replication-incompetent vectors that encode the histochemical reporter genes, lacZ and PLAP, at the ATCC in Rockville, Md. The
y2 and yCRE producers of BAG [18], a lacZ virus that we have used for lineage analysis, can be obtained by anyone and are listed as ATCC CRL 1858 (yCRE BAG) and 9560 (y2 BAG). Similarly, y2 producers of DAP [20], a vector encoding PLAP (described further below) is available as CRL 1949. For reasons that are unclear, the DAP line is more reliable for production of high titer stocks. Both of these vectors transcribe the reporter gene from the Mo-MLV LTR promoter and are generally useful for expression of the reporter gene in most tissues (though see discussion of promoters above). For lineage applications it is usually necessary to concentrate virus in order to achieve sufficient titer. This is typically due to a limitation in the volume that can be injected at any one site. Viruses can be concentrated fairly easily by a relatively short centrifugation step. Virions also can be precipitated using polyethylene glycol or ammonium sulfate, and the resulting precipitate collected by centrifugation. Finally, the viral supernatant can be concentrated by centrifugation through a filter that allows only small molecules to pass (e.g. Centricon filters). Regardless of the protocols used, one must keep in mind that the murine ecotropic and amphotropic retroviral particles, as well as the avian retroviral particles, are fragile, with short half-lives even under optimum conditions. In contrast, if one uses the VSV G protein as the viral envelope protein, the virions are more stable. In order to prepare the highest titered stock for multiple experiments, we usually concentrate several hundred milliliters of producer cell supernatant. These concentrated stocks are titered and tested for helper virus contamination, and can be stored indefinitely at -80°C or in liquid N2 in small (10- to 50-ml) aliquots. If we anticipate storage of over several months, we place a drop of mineral oil (e.g. a fresh tube of PCR oil) on top of the stock to prevent dehydration.REPLICATION-COMPETENT HELPER VIRUS
Replication-competent virus is sometimes referred to as helper virus as it can complement ("help") a replication-incompetent virus and thus allow it to spread from cell to cell. It can be present in an animal through exogenous infection (e.g. from a viremic animal in the mouse colony), expression of an endogenous retroviral genome (e.g. the akv loci in AKR mice), or through recombination events in an infected cell that occurs between 2 viral RNAs encapsidated in retroviral virions produced during packaging. The presence of helper virus is an issue of concern when using replication-incompetent viruses for lineage analysis as it can lead to horizontal spread of the marker virus, creating false lineage relationships. The most likely source of helper virus is the viral stock used for lineage analysis. The genome(s) that supplies the gag, pol, and env genes in packaging lines does not encode the
y sequence, but can still become packaged, although at a low frequency. If it is co-encapsidated with a vector genome, recombination in the next cycle of reverse transcription can occur. If the recombination allows the y- genome to acquire the y sequence from the vector genome, a recombinant that is capable of autonomous replication is the result. This recombinant can spread through the entire culture (although slowly due to envelope interference). Once this occurs, it is best to discard the producer clone or stock as there is no convenient way to eliminate the helper virus. As would be expected, recombination giving rise to helper virus occurs with greater frequency in stocks with high titer, with vectors that have retained more of the wild type sequences (i.e. the more homology between the vector and packaging genomes, the more opportunity there is for recombination), and within stocks generated using gagpol and env on a complementing genome during transfection (as opposed to 2 separate genomes, one for gagpol and one for env). Note that a murine helper genome itself will not encode a histochemical marker gene as apparently there is not room, or flexibility, within murine viruses that allows them to be both replication-competent and capable of expressing another gene like lacZ. The way that spread would occur is by a cell being infected with both the lacZ virus and a helper virus. Such a doubly-infected cell would then produce both viruses.When performing lineage analysis, there are several signs that can indicate the presence of helper virus within an individual animal. If one allows an animal to survive for long periods of time after inoculation, particularly if embryos or neonates are infected, the animal is likely to acquire a tumor when helper virus is present. Most naturally occurring replication-competent viruses are leukemogenic, with the disease spectrum being at least in part a property of the viral LTR. Secondly, if one analyzes either short or long term after inoculation, the clone size, clone number, and spectrum of labelled cells may be indicative of helper virus. For example, the eye of a newborn rat or mouse has mitotic progenitors for retinal neurons, as well as mitotic progenitors for astrocytes and endothelial cells. By targeting the infection to the area of progenitors for retinal neurons, we only rarely see infection of a few blood vessels or astrocytes as their progenitors are outside of the immediate area that is inoculated and they only get infected by leakage of the viral inoculum from the targeted area. However, if helper virus were present, we would see infection of a high percentage of astrocytes, blood vessels, and eventually, other eye tissues since virus spread would eventually lead to infection of cells outside of the targeted area. One would expect to see a correlation between the % of such non-targeted cells that are infected and the degree to which their progenitors are mitotically active after inoculation, due to the fact that infection requires a mitotic target cell. If one were to examine tissues other than ocular tissues, one would similarly see evidence of virus spread to cells whose progenitors would be mitotically active during the period of virus spread. In addition, the size and number of "clones" may also appear to be too large for true "clonal" events if helper virus were present. This interpretation of course relies upon some knowledge of the area under study.
DETERMINATION OF SIBLING RELATIONSHIPS
When performing lineage analysis, it is critical to unambiguously define cells as descendants of the same progenitor. This can be relatively straightforward when sibling cells remain rather tightly, and reproducibly, grouped. An example of such a straightforward case is the rodent retina, where the descendants of a single progenitor migrate to form a coherent radial array [31, 32]. The 2 analyses described below were applied to the rodent retina, and are applicable in any system where clones are arranged simply and reproducibly. The first assay is to perform a standard virological titration in which a particular viral inoculum is serially diluted and applied to tissue. In the retina, the number of radial arrays, their average size, and their cellular composition were analyzed in a series of animals infected with dilutions that covered a 3 log range. The number of arrays was found to be linearly related to the inoculum size, while the size and composition were unchanged. Such results indicate that the working definition of a clone, in this case a radial array, fulfilled the statistical criteria expected of a single hit event.
The second assay is to perform a mixed infection using 2 different retroviruses in which the histochemical reporter genes are distinctive. Two such viruses might encode cytoplasmically localized vs. nuclear-localized
b-gal. This can work when the cytoplasmically-localized b-gal is easily distinguished from the nuclear-localized b-gal [19, 33]. We have found that this is not the case in rodent nervous system cells as the cytoplasmically-localized b-gal quite often is restricted to neuronal cell bodies and is therefore difficult to distinguish from nuclear localized b-gal. In order to overcome this problem, we created the afore mentioned DAP virus [20], which is distinctive from the lacZ-encoding BAG virus. A stock containing BAG and DAP was produced by y2 producer cells grown on the same dish. The resulting supernatant was concentrated and used to infect rodent retina. The tissue was then analyzed histochemically for the presence of blue (due to BAG infection) and purple (due to DAP infection) radial arrays. If radial arrays were truly clonal, then each one should be only 1 color. Analysis of approximately 1100 arrays indicated that most were clonal. However, 5 comprised both blue and purple cells. This value will be an underestimate of the true frequency of incorrect assignment of clonal boundaries as sometimes 2 BAG or 2 DAP virions will infect adjacent cells and thus not lead to formation of bi-colored arrays. A closer approximation of the true frequency can be obtained by using the following formula (for derivation, see Fields-Berry et al. 1992 [20]).____________________________________________ = % errors
# total arrays
where a and b are the relative titers comprising the virus stock. The relative titer of BAG and DAPused in the co-infection was 3:1 and thus the value for percent errors in clonal assignments was 1.2%.
The value of 1.2% for errors in assignment of clonal boundaries includes errors due to both aggregation and independent virions (e.g. perhaps due to helper-virus mediated spread) infecting adjacent progenitors. The percent errors in other areas of an animal will depend upon the particular circumstances of the injection site, and upon the multiplicity of infection (MOI, the ratio of infectious virions to target cells). Most of the time MOI will be quite low (e.g. in the retina it was approximately 0.01 at the highest concentration of virus injected). Concerning the injection site, injection into a lumen, such as the lateral ventricles, should not promote aggregation nor high local MOI, but injection into solid tissue in which the majority of the inoculum has access to a limited number of cells at the inoculation site, could present problems. By co-injecting BAG and DAP, one can monitor the frequency of these events and thus determine if clonal analysis is feasible.
An error rate as small as 1.2% does not affect the interpretation of "clones" that are frequently found in a large data set. However, as with any experimental procedure that relies in some way on statistical analysis, rare associations of cell types must be interpreted with some caution and conclusions cannot be drawn independently of other data.
The above analysis was performed using viruses that were produced on the same dish and concentrated together. This was done as we felt that the most likely way that 2 adjacent progenitors might become infected would be through small aggregates of virions. Aggregation most likely occurs during the concentration step as one often can see macroscopic aggregates after resuspending pellets of virions. Thus, when the 2-marker approach is used to analyze clonal relationships, it is best to co-concentrate the 2 vectors together in order for the assay to be sensitive to aggregation due to this aspect of the procedure. (Although aggregation of virions may frequently occur during concentration, it apparently does not frequently lead to problems in lineage analysis presumably due to the high ratio of non-infectious particles to infectious particles found in most retrovirus stocks. It is estimated that only 0.1—1.0 % of the particles will generate a successful infection. Moreover, most aggregates are probably not efficient as infectious units; it must be difficult for the rare infectious particle(s) within such a clump to gain access to the viral receptors on a target cell.)
