For infection of non-rodent species, an envelope glycoprotein other than the ecotropic glycoprotein must be used to allow entry into the host cells. The one that endows the greatest host range is the VSV G glycoprotein, which allows infection of most species, including fish [11]. The G protein apparently also makes for a more stable particle, which allows for greater concentration of the virus preparations. For lineage analysis of avian species, packaging lines and vectors based upon avian retroviruses are available [12-14]. In addition, we recently found that avian retroviruses with the VSV G protein on their surface were more efficient at infecting chick embryos Figure 1 and [15]. Such virions gave the same titer as those with the avian A type env protein when they were titered on avian cells in vitro. However, when injected in vivo, the VSV G carrying particles give an approximately 350 fold more efficient infection, as judged by the number of clones in the retina, than the particles carrying the avian A env protein. Similar increases in efficiencies were noted throughout the embryo. We interpret these data to mean that cells within the avian embryo do not express high enough levels of the receptor for the A type env to be readily infected, but are not limited with respect to the ubiquitous phospholipid receptor for the VSV G protein [16]. We did not see this effect of increased efficiency of infection using murine vectors and murine embryos, but it is possible that different mouse strains vary in this regard. Gaiano et al. found that murine virions carrying VSV G gave a slightly different spectrum of cell types following infection of the early mouse brain than virions carrying the murine ecotropic env [17].
Figure 1:The VSV G protein endows avian retroviral vectors with a high efficiency of infection in vivo. Comparable amounts of an avian replication-incompetent vector, RIA-AP (ref. 15), encoding PLAP were injected into developing chick embryos. RIA-AP (G) virion particles contained VSV G protein on their surface and RIA-AP (A) virions contained the avian retroviral A env protein on their surface. Embryos were injected at stage 18 into the eye (A and B), stage 10 into the neural tube (E and F), or stage 18 into the limb bud or heart regions (C, D, G, H). Embryos injected with RIA-AP (A) are shown in panels A, C, E., and those injected with RIA-AP (G) in panels B, F, D, and H. Sections of infected limbs are shown in G and H. Approximately 3 days postinfection, the embryos were stained to reveal PLAP activity; red arrowheads indicate the limb regions and blue arrowheads the heart.
Two other parameters to be considered when choosing a vector for lineage analysis are the reporter gene and the promoter that drives its expression. The reporters that have been used include cytoplasmic lacZ [18], nuclear lacZ [19], human placental alkaline phosphatase (PLAP) [20], and avian gag [21]. More recently, there are vectors encoding green fluorescent protein (GFP) [22]. We have found advantages and disadvantages in the use of each of these reporters. When deciding which reporter to use, we first consider the background activities in the tissue of interest. Although the lacZ gene gives a stable, reliable, and specific signal in most cells using Xgal detection [23], there is problematic b-galactosidase background in a few tissues. Control staining with Xgal thus should be done to determine if it is a problem in an area of interest. Changes in the fixation and staining conditions can reduce b-galactosidase background [24]. Similarly, PLAP staining is reliable and stable, with heat treatment of the infected tissue rendering most endogenous alkaline phosphatases inactive. However, in some tissues residual alkaline phosphatases cause a backgroud problem. In such cases, inhibitors of endogenous alkaline phosphatases may solve the problem [25]. When using GFP, the signal from the introduced GFP can be weak, and thus background fluorescence in the tissue can be a problem. We have found that tissue sections prepared on a vibratome yield bright GFP+ cells, but the same tissue sectioned on a cryostat gives much dimmer and more ill-defined GFP+ cells.
The second issue to consider is how one wishes to define the cell types expressing the reporter gene. In some cases, the morphology of the infected cells indicates their identity. In such cases, we recommend that one use lacZ or PLAP with histochemical detection, which is the simplest and most rapid way to find the infected cells. Moreover, the Xgal precipitate formed by b-galactosidase and the XP/NBT product of PLAP are stable for months of storage, allowing one time to analyze many sections. However, there are differences between lacZ and PLAP that will direct your choice of which one to use for morphological identification. LacZ typically does not fill the cell bodies of large cells, such as neurons, as completely as PLAP. Thus, when it is desirable to characterize cells via their morphology, PLAP, which associates with the plasma membrane, is superior to lacZ. PLAP is also the most sensitive of the reporters that we have used, most likely due to the fact that it is a very stable enzyme. However, PLAP can produce such a dense stain that, when clonally-related cells are close together, we have been unable to count the number of cells in a clone or distinguish the morphologies of individual cells (see [26] for an example). In such cases, nuclear lacZ is very useful.
If one cannot use morphological criteria to identify the types of cells carrying the reporter gene, one option is to use immunohistochemistry to detect defining cellular antigens. The Xgal product of lacZ and the reaction product of PLAP make it difficult to detect a fluorescent immunohistochemical product as they absorb fluorescence. Moreover, the Xgal product and the X-P/NBT precipitate produced by PLAP often are too dark to allow simultaneous detection of another colored precipitate produced by immunohistochemical detection of a cellular antigen. However, occasionally, this will work (e.g. see [15, 27]). One can use double immunohistochemical procedures by employing antisera to detect PLAP or lacZ. However, double immunohistochemical procedures are much more time consuming then histochemistal procedures and immunohistochemical detection of lacZ and PLAP is not always as sensitive as the histochemical procedures. For all of these reasons, GFP is a better choice. GFP allows simultaneous detection of the GFP reporter and an immunohistochemical signal e.g. see [28]. However, as mentioned above, GFP expression is sometimes weak and, in addition, storage of sections over a long period of time (i.e. months) does not allow for preservation of the GFP signal. The newest reporter to be described, b-lactamase [29], may offer advantages over GFP in terms of sensitivity, but it has not yet been tested in vivo, where it may suffer from leakage of the product from infected cells. If this is a problem, future substrates might overcome this limitation.
The choice of the promoter to drive expression of a reporter gene also requires consideration. In order to see all the progeny of infected cells, a constitutive promoter should be used. We have had success using the LTR of Moloney Murine Leukemia Virus (Mo-MLV) for work in rats and mice and the LTR of Avian Leukosis Viruus for work in chicks. We compared several alternative promoters located internal to the Mo-MLV LTR, in the context of a wild type LTR promoter and in the context of an LTR promoter with an enhancer deletion, using infections of murine tissue in vivo [30, 31]. The LTR promoter performed the best of several promoters tested, including the human histone 4, chicken b-actin, CMV immediate early, and the SV40 early promoters. However, Gaiano et al. [17] recently reported that the Mo-MLV promoter was relatively inactive in early (E8.5 to E9.5) progenitor cells of the CNS. This problem is reminiscent of the failure of the LTR to express in embryonic stem cells and preimplantation embryos, which appears to be due to inhibition of the LTR in stem cells. Gaiano et al. found that an internal promoter of Ef1a or CMV/b-actin resulted in more expression in early progenitor cells as well as in later neurons. These findings suggest that one should test for stable expression in the area of interest, using the infection time and site that will be used for future experiments, before choosing the promoter. However, even after performing such preliminary experiments, and even with the choice of an apparently constitutive promoter, it is important to restrict one's conclusions about lineal relationships to cells that are marked and not to make assumptions about their relationships to cells that are unmarked. We have found, in some clones, that not all cells express a reporter gene. This is true even in control situations, such as in clones of NIH-3T3 fibroblasts infected with either a lacZ or PLAP vector in vitro. This observation has been made using several different promoters and vector designs.
