We find that outgrowth cells from bitransgenic cardiac explants can be c-kit+ but GFP− (Fig. 5), further negating an obligatory linkage between c-kit expression and GFP positivity.
Since the pedigrees showed the X-linked inheritance mode, 48 polymorphic microsatellite genetic markers, spanning the entire X chromosome at the average interval of 5 cM (Linkage Mapping Set-HD5 kit, PE Biosystems, Foster City, CA, USA), were selected to scan the genomic DNA extracted from peripheral blood leukocyte of Family A members.
For each sample, 2.5 μg of DNA was labeled using the Universal Linkage System (ULS) labeling kit (Kreatech Biotechnology) according to the manufacturer's instructions with minor modifications: 1 μL of Cy-ULS dye was added per 1 μg of genomic DNA adjusted with 10× labeling buffer.
PDGFRA is not mapped in the cat; however, a KIT-PDGFRA linkage group is found in several mammalian species.
PDGFRA has not been localized to feline chromosome B1; however, the KIT-PDGFRA linkage group is highly conserved in other species.
Plasmid DNA was labeled with Alexa Fluor 488 or 594 by Universal Linkage System (ULS™) using the ULYSIS nucleic acid labeling kit (Invitrogen, Molecular Probes, Inc., Carlsbad, CA) with modifications as described by Coelho-Castelo et al [50].
Testing for linkage in families of different breeds could provide additional evidence for KIT or PDGFRA as candidate genes for white spotting in cats.
Significant linkage to W was established with three STRs tightly linked to the feline KIT locus on chromosome B1 (θ=0, LOD= 6.0 6.3) (Table 1).
The effective use of TBAs should incorporate training, linkage to health care services, continuous in-service training and skills development, access to clean delivery kits, resuscitation equipment and effective referral pathways (Sibley et al. 2007; Wilson et al. 2011).
