Detecting fa leptin receptor mutation in Zucker rats with tetra-primer amplification-refractory mutation system (ARMS)-PCR

Abstract

Due to the genetic mutation (fa) in the gene encoding for leptin receptor, homozygous Zucker rats (fa^-/-) develop excessive adiposity and become an experimental animal model in obesity and metabolic-related diseases research. Based on tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), we developed a method to quickly genotype Zucker rats with a mutated fa allele from their wildtype littermates. The three genotypes are clearly discriminated on 2.0% agarose gel. Our method can be used as a reliable tool to set up and maintain the breeding colony in animal facilities as well as assign animals to control and treatment groups based on their genotypes for animal studies.

Keywords: Leptin receptor; Obesity; Tetra-primer amplification-refractory mutation system (ARMS); Zucker rats; fa mutation.

Fig. 1

Schematic delineation of DNA amplicon patterns on the agarose gel. FO and RO primers are annealed at positions located at varying distances from the fa mutation site (framed in box) to generate different sizes of allele-specific PCR amplicons. To increase the specificity of the PCR reaction, base “C” (underlined and red-colored) in FI primer (purple) was purposely introduced at position −2 from 3 prime end.

Fig. 2

Results of gradient PCR reaction with the introduction of second mismatch nucleotide at position-2 from the 3-prime end of the RI primer. As shown in the figure, although tetra-primer ARMS-PCR successfully discriminates the different genotype patterns on the gel, the introduction of second point mutation (5′-GATTGTAGAATTCTCTAAATATTTCATCG-3′, “T”, in red) in the RI primer, significantly reduced the efficiency of DreamTaq to generate amplicons from heterozygous (fa^+/−) rat genomic DNA as indicated by the low density of the 263 bp band. fa^+/+: wildtype; fa^+/−: heterozygous, and fa^−/− homozygous Zucker rats. Different sizes of PCR amplicons were indicated by red arrows.

Fig. 3

Patterns of amplicons generated from the tetra-primer ARMS-PCR reaction on agarose gel. DNA samples collected from three Zucker rats with known genotypes (wildtype (fa^+/+), heterozygous (fa^+/−), and homozygous (fa^−/−) were analyzed in a gradient PCR (annealing temperatures from 48.3 to 64.9 °C) to identify an optimal annealing temperature for the assay on the 2.0% agarose gel. Different sizes of PCR amplicons were indicated by red arrows.

Fig. 4

Identifying genotypes of Zucker rats. Fig. 4a: Genome DNA collected from the livers of rats were amplified, followed by gel electrophoresis and visualized on 2.0% agarose gel. Each lane represents an individual Zucker rat. Fig. 4b: The genotypes of the same 12 Zucker rats were examined with PCR-RFLP after Pvu II digestion on a 4.0% agarose gel. Each lane represents an individual Zucker rat. The red arrows represent the size of amplicons on the gel.

Fig. 5

The changes in body weights of Zucker rats over time. The Zucker rats' body weights were measured between the ages of 3–7 weeks. For each group, data were presented as mean ± SEM: fa^+/+ (n = 2), fa^+/− (n = 4), and fa^−/− (n = 6). Statistical analysis was conducted with two-way ANOVA followed by Student-Newman-Keuls post hoc test. The fa^−/− homozygous rats were substantially heavier than the wildtype (fa^+/+) and heterozygous (fa^+/−) rats between the age of 6 and 7 weeks. A significant level was set at p < 0.05.

Fig. 6

Sequencing results of amplicons of the tetra-primer ARMS-PCR reaction. PCR amplicons representing wildtype, heterozygous, and fa^−/− alleles were extracted from the agarose gel and sequenced. Fig. 6a shows a representative sequence of the 201 bp band (A allele, wildtype); Fig. 6b shows a representative sequence of the 263 bp band, which contained an A to C point mutation.