Verification of Equine Keratinocyte Cell Lines

Additional verification that the primary keratinocyte cultures EK1547 and EK100 were of pure equine origin and not mixed with other cell lines was done by FISH. We used horse bacterial artificial chromosome (BAC) clones (CHORI-241: http://bacpac.chori.org/equine241.htm) containing select chromosome-specific markers (Table 2). BAC DNA was isolated by Plasmid Midiprep kit (Qiagen), labeled with biotin-16-dUTP or digoxigenin-11-dUTP using Biotin- or DIG-Nick Translation Mix (Roche, Basel, Switzerland), and hybridized to metaphase chromosomes. Hybridizations and signal detection were carried out according to standard protocol [25 (link)]. The results were examined with Zeiss Axioplan2 fluorescence microscope and at least ten images were captured and analyzed for each experiment using Isis V5.2 (MetaSystems GmbH) software.Table 2

Horse chromosome specific BAC clones used for FISH

BAC ID	Horse chromosome	Known gene content	Reference	Label and detection
049H16	9	LCORL	Staiger et al. 2016 [35 (link)]	Biotin-FITC
076H13	29	CREM	Ghosh et al. 2014 [36 (link)]	Digoxigenin-Rhodamine

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Alkhilaiwi F., Wang L., Zhou D., Raudsepp T., Ghosh S., Paul S., Palechor-Ceron N., Brandt S., Luff J., Liu X., Schlegel R, & Yuan H. (2018). Long-term expansion of primary equine keratinocytes that maintain the ability to differentiate into stratified epidermis. Stem Cell Research & Therapy, 9, 181.

Publication 2018

Plasmid Midiprep kit DIG-Nick Translation Mix Isis V5.2

Bacterial artificial chromosome Biotin Biotin 16 dutp Cell lines Chromosome Chromosome markers Clones Digoxigenin Digoxigenin 11 dutp Equine Fish Fluorescence microscope Gene Horse Hybridizations Keratinocyte Metaphase Plasmid Rhodamine Signal detection

Corresponding Organization : Georgetown University

Other organizations : Texas A&M University, University of Veterinary Medicine Vienna, North Carolina State University

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Variable analysis

independent variables

Horse bacterial artificial chromosome (BAC) clones (CHORI-241) containing select chromosome-specific markers

dependent variables

Presence and location of chromosome-specific markers on metaphase chromosomes

control variables

Metaphase chromosomes

controls

No positive or negative controls were explicitly mentioned.

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