*2.2. Vectors*

StarTrack constructs were designed as described previously [16,17], and di fferent combinations of StarTrack constructs were used separately to target the di fferent profiles of the progenitor cells. The hyperactive transposase of the PiggyBac system (CMV-hyPBase) was used to generate di fferent vectors in which the expression of the transposases was driven by promoters for NG2, GFAP, and GSX2. The cloning of the di fferent hyPBase constructs was performed by Canvax Biotech, and the source of the promoters is indicated in Table 1. All plasmids were sequenced (Sigma–Aldrich; Saint Louis, MO, USA) to confirm successful cloning. This strategy allowed specific progenitors with active gene expression of these promoters at the time of electroporation to be labeled in order to track their full progeny. Plasmid mixtures contained the twelve UbC-StarTrack floxed constructs, a transposase of the PiggyBac system under the control of the selected specific promoter (either CMV, NG2, GFAP or Gsx2), and the CAG-CreERT2 vector to remove the episomal copies of constructs [17].


**Table 1.** List of the di fferent plasmids used in the StarTrack approach

#### *2.3. In Utero Electroporation (IUE) and Tamoxifen Administration*

In utero electroporation was performed as described previously [17,27]. Briefly, the selected plasmid mixture was injected into the LV of E12 embryos with a micropipette and using an ultrasound device (VeVo-770; VisualSonics, Toronto, Canada) and then co-electroporated (three animals per experimental group). The embryos were returned to the dam's abdominal cavity, which were then monitored for three days. After birth, all the pups were injected with tamoxifen (Tx, 20 mg/ml dissolved in pre-warmed corn oil: Sigma–Aldrich) to eliminate episomal copies of the plasmids and to achieve heritable and stable labelling of the cell progeny [17]. A single dose of Tx (5 mg/40 gr body weight) was administered intraperitoneally (i.p.) to the litter at P5. The mice were analyzed from P30 onwards.

## *2.4. Tissue Processing*

All mice were analyzed at adult stages, anesthetizing them with an i.p. injection of pentobarbital (Dolethal 40–50 mg/Kg; Vetoquinol, Alcobendas, Madrid) and then perfused with 4% paraformaldehyde (PFA) in 0.1M phosphate bu ffer (PB). Subsequently, the brain of mice was removed and postfixed for two hours in fresh 4% PFA and then in PB. Coronal vibratome sections of the brains (50 μm) were obtained and mounted onto a glass slide with Mowiol for storage at 4 ºC.
