For metaphase spread preparation, mitoses were mechanically collected by blowing the medium on the dish surface. Chromosome preparations were performed with the standard air-drying procedure and were used for karyotype analysis or fluorescence in situ hybridization (FISH).
Whole genomic DNA from Przewalski’s horse fibroblasts was extracted according to standard procedures. BAC clones (CHOR1241-17G8, coordinates in EquCab2.0: chr5:37,011,456–37,167,308; CHOR1241-25H7 coordinates in EquCab2.0: chr5: 98,031,303–98,224,666) and lambda phage 37cen and 2PI clones [16 (link),53 (link)] were extracted from 10 mL bacterial cultures with the Quantum Prep Plasmid miniprep kit (BioRad, Milan, Italy), according to supplier instructions. All probes were labeled by nick translation with Cy3-dUTP (Enzo Life Sciences, Milan, Italy) or Alexa488-dUTP (Life Technologies, Monza, Italy) as previously described [64 (link)]. FISH was performed as previously described [68 (link)].
Chromosomes were counterstained by DAPI. Digital grayscale images for fluorescence signals were acquired with a fluorescence microscope (Zeiss Axio Scope.A1, Zeiss, Göttingen, Germany) equipped with a cooled CCD camera (Teledyne Photometrics, Birmingham, UK). Pseudocoloring and merging of images were performed using the IPLab 3.5.5 Imaging Software (Scanalytics Inc., Fairfax, VA, USA). Chromosomes were identified by DAPI banding according to the published karyotypes [49 (link),50 (link)].
Whole genomic DNA from Przewalski’s horse fibroblasts was extracted according to standard procedures. BAC clones (CHOR1241-17G8, coordinates in EquCab2.0: chr5:37,011,456–37,167,308; CHOR1241-25H7 coordinates in EquCab2.0: chr5: 98,031,303–98,224,666) and lambda phage 37cen and 2PI clones [16 (link),53 (link)] were extracted from 10 mL bacterial cultures with the Quantum Prep Plasmid miniprep kit (BioRad, Milan, Italy), according to supplier instructions. All probes were labeled by nick translation with Cy3-dUTP (Enzo Life Sciences, Milan, Italy) or Alexa488-dUTP (Life Technologies, Monza, Italy) as previously described [64 (link)]. FISH was performed as previously described [68 (link)].
Chromosomes were counterstained by DAPI. Digital grayscale images for fluorescence signals were acquired with a fluorescence microscope (Zeiss Axio Scope.A1, Zeiss, Göttingen, Germany) equipped with a cooled CCD camera (Teledyne Photometrics, Birmingham, UK). Pseudocoloring and merging of images were performed using the IPLab 3.5.5 Imaging Software (Scanalytics Inc., Fairfax, VA, USA). Chromosomes were identified by DAPI banding according to the published karyotypes [49 (link),50 (link)].
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