Here we have characterized two independent transgenic mouse lines in which Cre recombinase is expressed under the control of the mouse Plp
promoter. We demonstrate the Plp
promoter cassette's capability to drive expression of a Cre recombinase transgene within the CNS, but in a more widespread pattern than initially anticipated. This was particularly true at early developmental stages when the transgene was expressed in neural tissues, and not restricted to the oligodendrocyte lineage, results corroborated by the work of others 
. During this time, transgene expression was also localized to the PNS (sciatic nerve, ventral and dorsal roots), most likely in Schwann cells. It was only at late postnatal stages that the Plp-Cre
transgene demonstrated a high level of oligodendrocyte specificity.
At the mRNA level, Cre expression was highly restricted to CNS tissues (within those examined) at early and late stages of postnatal development. At both P4 and P28, transgene expression was detected in cortex, cerebellum, and spinal cord. In addition, at P4, Cre transcript was also detected in the kidney, liver, skeletal muscle, and heart of mice from line 627, albeit at a level lower than that in neural tissues.
At the protein level in the optic nerve, spinal cord, and cerebellum of P28 transgenic mice Cre recombinase was predominantly in mature oligodendrocytes as identified by CC-1 staining. However, at P4, Cre protein expression was more widespread, localizing to granular () and Purkinje cell layers within the cerebellum, and throughout spinal cord astrocytes. Previous studies have also identified Plp
gene products in the external granular and Purkinje cell layers of the developing mouse cerebellum, as well as in grey matter astrocytes of the developing spinal cord 
The fact that the Plp-CreERt transgenic mice have an inducible Cre protein allowed for tracking of the Plp promoter activity from specific developmental stages onwards. Indeed, the administration of tamoxifen at specific time points allowed us to follow the fate of cells that were Plp positive at the time of administration. The reporter-positive cells observed at later stages are the progeny of Plp promoter active cells at the time of tamoxifen administration. Mice given tamoxifen at P4 displayed the Plp promoter's promiscuity at this early time point, with the result that β-galactosidase reporter expression was detected in neuronal non-white matter regions at later time points. In stark contrast, the β-galactosidase expression profile in mice receiving tamoxifen at P16 was limited strictly to white matter zones at later stages of analysis. From these studies, a clear Plp promoter driven spatio-temporal patterning emerges for the CNS – that of neuronal inclusion early in postnatal development, with later stages defined by increased oligodendrocyte specificity.
Our transgene product, by analysis of Plp-Cre;Rosa26LacZ mice, proved to be functional in a large range of cell types during embryogenesis. This is dramatically demonstrated in the crosses to the mT/mG reporter mice, in which, by P28 all but discreet zones of CNS vasculature expressed excision activated EGFP.
To better understand where and when the Plp
promoter is active in the CNS, it is important to consider the products of the Plp
gene. The promoter drives production of both PLP and its splice variant DM-20. The splice site is located within exon three of the gene, producing a transcript product lacking exon 3B 
. The protein structure of the smaller isoform DM-20 is identical to PLP except for the removal of amino acids 116 through 150 
. DM-20 mRNA has been detected by RT-PCR in the murine neural tube and spinal cord during embryonic development, whereas the PLP transcript is the predominant postnatal isoform 
. In situ
hybridization experiments at E10 using a DM-20 anti-sense probe detected signal in both the CNS (neural tube within the diencephalic basal plate) and the PNS (trigeminal and spinal ganglia, and sympathetic ganglion chain) 
. Other studies have confirmed this observation, demonstrating the presence of PLP/DM-20 RNA transcript as early as E9.5 
. DM-20 is therefore clearly present in cells long before the appearance of defined mature or even precursor oligodendrocytes. Previous studies have shown DM-20 outside of the glial cell lineage, with expression detected in diverse cell types, including various neuronal cell lines such as G-26, B104, NG108-15, NG18-TG, Neuro2A, PC12, and P19 
. PLP/DM-20 transcripts and/or protein have also been found in cells of the thymus, spleen and testicles 
Recently, various groups have identified specific cellular subtypes within the murine CNS expressing Plp
gene products at early embryonic and postnatal stages. One such study tracked the fate of NG2+
oligodendrocyte progenitors co-labeling for Plp
promoter driven EGFP 
. They demonstrated the ability of the progenitors to not only differentiate into myelinating oligodendrocytes, but also into astrocytes and neurons. Another study compared PLP/DM-20 transcript levels at P5 and adult stages, between whole murine cortex and isolated NG2-positive cells. PLP mRNA was detected in isolated NG2-positive cells at both time points, whereas DM-20 mRNA was amplified from whole cortex only 
. Similar to our own experiments, Delauney and colleagues crossed Plp-Cre
mice to a GFP reporter, subsequently identifying GFP-positive neuroepithelial cells at E9.5, with the expressing profile switching to reporter-positive radial glial cells by E13.5 
Thus, it is clear that the two Plp gene products yield varying and different expression profiles, and can be generally classified as follows – Plp, while expressed early in various CNS cellular subtypes, is at its highest levels postnatally and in an oligodendrocyte-specific manner, while DM-20 is preferentially expressed early and diffusely in the embryo. It is therefore important to recognize the possibility for Plp promoter driven transgene expression simply as a by-product through activation of factors normally involved in DM-20 regulation.
Interestingly, in 1999 a novel exon was discovered lying within intron 1 of the Plp
gene. Termed exon 1.1, it produced a protein with an additional 12 amino acid leader sequence 
. The additional sequence allowed for expression of two further Plp
variants, termed sr-PLP and sr-DM-20. These proteins have been detected in oligodendrocytes, muscle, and to an even larger extent in neurons 
. Two similar novel exons have recently been discovered in intron 1 of the human Plp1
gene, giving rise to unique PLP isoforms 
. The isoforms are expressed predominantly in neurons and to lesser extent in oligodendrocytes, beginning during human fetal development. The authors suggest a role for the proteins in axonal-glial communication, the disruption of which would account for neuronal dysfunction in humans carrying Plp1
The very early and widespread expression of our transgene product forces us to reconsider certain aspects of Plp
gene regulation. Perhaps there are regulatory elements activated differentially for each of the Plp
derived isoforms, responsible for the specific spatial and temporal expression patterns observed during embryonic and postnatal development. Recent evidence suggests that this could indeed be the case. Within the Plp
gene, six evolutionary conserved non-protein coding sequences were identified as Plp/DM-20
enhancers, five of which were located within intron 1 
. When inserted as single copies driving expression of an EGFPLacZ
reporter gene, each unique regulatory sequence matched characterized components of the Plp
promoter's spatio-temporal expression profile as seen through a spectra of glial and neuronal lineage patterning.
The present study informs on Plp promoter functionality as a driver for both Cre and other transgene expression. Since the goal of many such studies is oligodendrocyte targeting within the CNS, it is important to better understand differences in gene regulation between Plp splice variants. Once more information is obtained deciphering the different regulatory elements governing expression of Plp/DM-20 and sr-Plp/sr-DM-20, one could perhaps design a more specific promoter construct, which would allow for a postnatal, and more robust oligodendrocyte-specific expression of the requisite transgene. In the mean time, our work would stress that as long as one uses the tamoxifen inducible system (Plp-CreERt) and induces Cre activity at adult stages, one can achieve oligodendrocyte specific excision of target genes.