Isolation, sequencing and relative quantitation by fluorescent-ratio PCR of feline β-lactoglobulin I, II, and III cDNAs

Beta-lactoglobulin (βLG) is a whey protein found in the milk of most mammals, except those of humans and rodents. In ruminants, only one type of βLG is expressed in milk, although the presence of pseudogenes has been reported. The milk of other mammals (dog, horse, dolphins) contains two types of βLG: type I is related to ruminant βLG, while type II has a strong similarity to the ruminant pseudogenes. The presence of three types of βLG has been described only in cat milk. For the first time, we have cloned, sequenced, and characterized the three types of feline βLG cDNAs from a mammary gland sample of a lactating cat. Since no specific probe could be easily used to differentiate them by Northern Blotting, we developed a new technique named Fluorescent-Ratio PCR (FR-PCR) in order to assess their level of expression during lactation. The relative amounts of each feline βLG cDNA was quantified in two cats by capillary electrophoresis of the restricted RT- PCR product, labeled with fluorescent primers. We observed the same percentage of expression of the three βLG genes in two cats. The differences of expression could be due to changes in the promoter and 3' non-coding region affecting the level of transcription and the mRNA stability. The FR- PCR technique was shown to be reproducible and accurate even for small percentage differences, being very useful when a small amount of sample is available.