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Extra info for homeobox gene expression in adult dorsal root ganglia is regeneration a recapitulation of development christina francisca vogelaar
To follow up this screening, we aimed to quantify the expression of these genes during the regeneration process using real-time quantitative PCR. Because of the availability of mouse gene sequences in the databases and the potential to use mouse mutants as experimental animal in regeneration experiments, we switched to the mouse sciatic nerve crush model. 55 Chapter 4 Table 1: Homeobox gene expression in regenerating rat DRGs Homeobox gene DRG11 Gbx2 Gsc Gsh4 Hoxa1 Hoxc5 Lmx1b Msx1 Msx3-like Otp Pax3 Semi-q - Homeobox gene Semi-q Prx2 Prx3 Ptx2 Vsx2-like Zfh4 Brn2 Brn3a Brn3b Brn4/RHS2 Oct1 Oct6 - The homeobox genes identified using semiquantitative measurements in sham- and crushoperated rat DRGs.
Regarding the homeobox genes the positions of primers and probes were chosen such that the homeobox was not included in the PCR. 5 cDNA and products were sequenced to check the specificity. Products were cloned into pGEM-T easy vectors following the manufacturer’s protocol and colony PCRs were performed with primers directed to the T7 and SP6 sites in the vector. For optimization 104 copies of the purified colony PCR products were produced and used as templates to determine the optimal concentration of primers and probes (Table 2).
In situ hybridization For in situ hybridization, 3 mice received a crush lesion, 3 other mice were sham-operated. At 1, 4, and 7 dpo, ipsi- and contralateral DRGs corresponding to spinal level L5 were quickly dissected and cleaned from surrounding tissue. Per time point ipsi- and contralateral DRGs of both sham- and crush-operated animals were placed on a flat disc of frozen TissueTek, then covered with TissueTek and frozen on dry ice. Cryostat sections, cut at 8 µm thickness, were thaw-mounted onto SuperFrost®Plus slides (Menzel-Glaser), dried and stored at –80ºC.