Additional file 5: of Genetic dissection of the fatty liver QTL Fl1sa by using congenic mice and identification of candidate genes in the liver and epididymal fat

Allelle variants on chromosome 12 (Centromere-46.75 Mb) in SM/J strain versus C57BL/6 strain. (XLSX 13 kb

Additional file 1: of Detection of differentially expressed candidate genes for a fatty liver QTL on mouse chromosome 12

The primers for SYBR Green assay in real-time PCR. (DOCX 14 kb

Searching for Genomic Region of High-Fat Diet-Induced Type 2 Diabetes in Mouse Chromosome 2 by Analysis of Congenic Strains

<div><p>SMXA-5 mice are a high-fat diet-induced type 2 diabetes animal model established from non-diabetic SM/J and A/J mice. By using F2 intercross mice between SMXA-5 and SM/J mice under feeding with a high-fat diet, we previously mapped a major diabetogenic QTL (<i>T2dm2sa</i>) on chromosome 2. We then produced the congenic strain (SM.A-<i>T2dm2sa</i> (R0), 20.8–163.0 Mb) and demonstrated that the A/J allele of <i>T2dm2sa</i> impaired glucose tolerance and increased body weight and body mass index in the congenic strain compared to SM/J mice. We also showed that the combination of <i>T2dm2sa</i> and other diabetogenic loci was needed to develop the high-fat diet-induced type 2 diabetes. In this study, to narrow the potential genomic region containing the gene(s) responsible for <i>T2dm2sa</i>, we constructed R1 and R2 congenic strains. Both R1 (69.6–163.0 Mb) and R2 (20.8–128.2 Mb) congenic mice exhibited increases in body weight and abdominal fat weight and impaired glucose tolerance compared to SM/J mice. The R1 and R2 congenic analyses strongly suggested that the responsible genes existed in the overlapping genomic interval (69.6–128.2 Mb) between R1 and R2. In addition, studies using the newly established R1A congenic strain showed that the narrowed genomic region (69.6–75.4 Mb) affected not only obesity but also glucose tolerance. To search for candidate genes within the R1A genomic region, we performed exome sequencing analysis between SM/J and A/J mice and extracted 4 genes (<i>Itga6, Zak</i>, <i>Gpr155,</i> and <i>Mtx2</i>) with non-synonymous coding SNPs. These four genes might be candidate genes for type 2 diabetes caused by gene-gene interactions. This study indicated that one of the genes responsible for high-fat diet-induced diabetes exists in the 5.8 Mb genomic interval on mouse chromosome 2.</p></div

The genomic construct of congenic lines used to narrow <i>T2dm2sa</i> QTL location.

<p>The genome of each line has an SM/J background (white boxes) and was replaced by donor A/J genomic intervals (black boxes). Gray boxes show that it is not clear whether the genomic intervals were derived from A/J or from SM/J mice. Two bold solid lines indicate the genomic intervals of QTLs (<i>t2dm3</i> and <i>Nidd5</i>) for type 2 diabetes in BTBR mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096271#pone.0096271-Stoehr1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096271#pone.0096271-Stoehr2" target="_blank">[7]</a> and TOSD mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096271#pone.0096271-Hirayama1" target="_blank">[9]</a>, respectively. Two arrows indicate the genomic regions affecting the diabetic phenotypes, which were confirmed by using Moo-C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096271#pone.0096271-Stoehr2" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096271#pone.0096271-Karunakaran1" target="_blank">[8]</a> or Nidd5/3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096271#pone.0096271-Mizutani1" target="_blank">[10]</a> congenic strains. The dotted line indicates the genomic interval detected by meta-analysis for the diabetes-related QTLs of rodents <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096271#pone.0096271-Schmidt1" target="_blank">[12]</a>.</p

Body mass index, food intake, serum lipids, and tissue weights in SM/J, R0, and R1A congenic mice fed a HF diet for 11 weeks.

<p>TG, triglyceride; TC, total cholesterol.</p>ab<p>Means not sharing a common lowercase letter are significantly different by Tukey-Kramer’s test (<i>P</i><0.05).</p

The genomic construct of congenic lines used to narrow <i>T2dm2sa</i> QTL location.

<p>The genome of each line has an SM/J background (white boxes) and was replaced by donor A/J genomic intervals (black boxes). Gray boxes show that it is not clear whether the genomic intervals were derived from A/J or from SM/J mice. Two bold solid lines indicate the genomic intervals of QTLs (<i>t2dm3</i> and <i>Nidd5</i>) for type 2 diabetes in BTBR mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096271#pone.0096271-Stoehr1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096271#pone.0096271-Stoehr2" target="_blank">[7]</a> and TOSD mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096271#pone.0096271-Hirayama1" target="_blank">[9]</a>, respectively. Two arrows indicate the genomic regions affecting the diabetic phenotypes, which were confirmed by using Moo-C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096271#pone.0096271-Stoehr2" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096271#pone.0096271-Karunakaran1" target="_blank">[8]</a> or Nidd5/3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096271#pone.0096271-Mizutani1" target="_blank">[10]</a> congenic strains. The dotted line indicates the genomic interval detected by meta-analysis for the diabetes-related QTLs of rodents <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096271#pone.0096271-Schmidt1" target="_blank">[12]</a>.</p

The genes identified non-synonymous mutations in the R1A region (Chr. 2: 69.6–75.4 Mb) by exome sequencing.

a<p>AA means amino acid.</p>b<p>Mu (<i>Mus musculus</i> reference residue in C57BL/6), Rat (<i>Rattus norvegicus</i>), Ho (<i>Homo sapiens</i>), Bos (<i>Bos taurus</i>).</p

Diabetes-related traits in SM/J, original congenic R0, and congenic (R1, and R2) strains.

<p>A. The blood glucose concentrations and B. AUC during the glucose tolerance test, and C. serum insulin concentrations. ^abc,Means not sharing a common superscript letter were significantly different among SM/J, original congenic (R0), R1, and R2 congenic strains by Tukey-Kramer test.</p

DataSheet_1_FcRY is a key molecule controlling maternal blood IgY transfer to yolks during egg development in avian species.docx

Maternal immunoglobulin transfer plays a key role in conferring passive immunity to neonates. Maternal blood immunoglobulin Y (IgY) in avian species is transported to newly-hatched chicks in two steps: 1) IgY is transported from the maternal circulation to the yolk of maturing oocytes, 2) the IgY deposited in yolk is transported to the circulation of the embryo via the yolk sac membrane. An IgY-Fc receptor, FcRY, is involved in the second step, but the mechanism of the first step is still unclear. We determined whether FcRY was also the basis for maternal blood IgY transfer to the yolk in the first step during egg development. Immunohistochemistry revealed that FcRY was expressed in the capillary endothelial cells in the internal theca layer of the ovarian follicle. Substitution of the amino acid residue in Fc region of IgY substantially changed the transport efficiency of IgY into egg yolks when intravenously-injected into laying quail; the G365A mutant had a high transport efficiency, but the Y363A mutant lacked transport ability. Binding analyses of IgY mutants to FcRY indicated that the mutant with a high transport efficiency (G365A) had a strong binding activity to FcRY; the mutants with a low transport efficiency (G365D, N408A) had a weak binding activity to FcRY. One exception, the Y363A mutant had a remarkably strong binding affinity to FcRY, with a small dissociation rate. The injection of neutralizing FcRY antibodies in laying quail markedly reduced IgY uptake into egg yolks. The neutralization also showed that FcRY was engaged in prolongation of half-life of IgY in the blood; FcRY is therefore a multifunctional receptor that controls avian immunity. The pattern of the transport of the IgY mutants from the maternal blood to the egg yolk was found to be identical to that from the fertilized egg yolk to the newly-hatched chick blood circulation, via the yolk sac membrane. FcRY is therefore a critical IgY receptor that regulates the IgY uptake from the maternal blood circulation into the yolk of avian species, further indicating that the two steps of maternal–newly-hatched IgY transfer are controlled by a single receptor.</p