Science Department, Mary G. Montgomery High School, Semmes Alabama1;

Cellular and Molecular Science, Department of Animal Sciences, Auburn University, Auburn, Alabama2

Michael A. Plattenburg1, Gesa M. Capers1, and Terry D. Brandebourg2

Objectives:

Introduction:

Implications for Teaching / Learning:

Methodology:

Supported by National Science Foundation Grant NSF EPS-1158862

Alabama is at the center of an obesity epidemic that is causing increases in type 2 diabetes and heart failure. However, no effective strategies exist to prevent obesity. If we could better understand how obesity, diabetes, and heart failure are linked, it might be possible to create cures and thus prevent people from suffering these problems. To study obesity and diabetes, the Mangalica pig was imported to Auburn University for use as a biomedical model of juvenile obesity because these pigs demonstrate a very extreme, early onset, obese phenotype. The Mangalica or “wooly” pigs are native to Hungary and are curly haired and morbidly obese. Preliminary findings indicate that wooly pigs amass thick layers of adipose tissue which leads to insulin resistance and liver dysfunction. In humans these symptoms are associated with diabetes, heart disease, and other metabolic problems. In this regard, the wooly pig is a good model for these problems in humans. Concerning obesity, melanocortin receptors regulate food intake and play important roles in regulating body weight. One such receptor, the melanocortin 3 receptor (MC3R) signals the brain to stop eating. Mutations in this gene are known to directly cause obesity in rodents and humans. Given the known role for mutations in MC3R to cause obesity, it is possible that mutations in the wooly pig MC3R gene may in part explain the extreme obesity seen in these pigs. The aim of this TEA fellowship is to test this hypothesis by cloning the MC3R gene from wooly pig DNA and examining the gene for potential mutations that could alter the function of the melanocortin 3 receptor.

Alabama is at the center of an obesity epidemic that is causing increases in type 2 diabetes and heart failure. However, no effective strategies exist to prevent obesity. If we could better understand how obesity, diabetes, and heart failure are linked, it might be possible to create cures and thus prevent people from suffering these problems. To study obesity and diabetes, the Mangalica pig was imported to Auburn University for use as a biomedical model of juvenile obesity because these pigs demonstrate a very extreme, early onset, obese phenotype. The Mangalica or “wooly” pigs are native to Hungary and are curly haired and morbidly obese. Preliminary findings indicate that wooly pigs amass thick layers of adipose tissue which leads to insulin resistance and liver dysfunction. In humans these symptoms are associated with diabetes, heart disease, and other metabolic problems. In this regard, the wooly pig is a good model for these problems in humans. Concerning obesity, melanocortin receptors regulate food intake and play important roles in regulating body weight. One such receptor, the melanocortin 3 receptor (MC3R) signals the brain to stop eating. Mutations in this gene are known to directly cause obesity in rodents and humans. Given the known role for mutations in MC3R to cause obesity, it is possible that mutations in the wooly pig MC3R gene may in part explain the extreme obesity seen in these pigs. The aim of this TEA fellowship is to test this hypothesis by cloning the MC3R gene from wooly pig DNA and examining the gene for potential mutations that could alter the function of the melanocortin 3 receptor.

1. To successfully isolate genomic DNA and use conventional PCR to clone the MC3R gene from blood samples obtained from Mangalica pigs at Auburn University.

2. To successfully obtain DNA sequence information for the PCR products generated. 3. To utilize bioinformatics tools to determinate if sequence differences exist between the wild

type porcine MC3R sequence obtained from GenBank and sequences obtained from our cloned genes and if so, determine if such differences would lead to a protein with altered function.

1. To successfully isolate genomic DNA and use conventional PCR to clone the MC3R gene from blood samples obtained from Mangalica pigs at Auburn University.

2. To successfully obtain DNA sequence information for the PCR products generated. 3. To utilize bioinformatics tools to determinate if sequence differences exist between the wild

type porcine MC3R sequence obtained from GenBank and sequences obtained from our cloned genes and if so, determine if such differences would lead to a protein with altered function.

Results:Results:

Mary G. Montgomery HS will be the seat of a biomedical career academy and an ideal place to implement the procedures utilized in this study. PCR kits are readily available for laboratory use via Alabama Science in Motion. Students will be able to utilize the PCR equipment to run gel electrophoreses, identify genetic markers, and compare DNA ladders, genes, and AA sets for similarities and differences. Furthermore, a discussion regarding lifestyle, including healthy vs. unhealthy eating habits and choices, resulting in possible obesity, may ensue. In logical sequence this information will lead to investigations of onset, symptoms, diagnosis, and treatment of diseases related to obesity, such as diabetes type II, coronary heart disease, cardiac infarction, stroke, and others.

Mary G. Montgomery HS will be the seat of a biomedical career academy and an ideal place to implement the procedures utilized in this study. PCR kits are readily available for laboratory use via Alabama Science in Motion. Students will be able to utilize the PCR equipment to run gel electrophoreses, identify genetic markers, and compare DNA ladders, genes, and AA sets for similarities and differences. Furthermore, a discussion regarding lifestyle, including healthy vs. unhealthy eating habits and choices, resulting in possible obesity, may ensue. In logical sequence this information will lead to investigations of onset, symptoms, diagnosis, and treatment of diseases related to obesity, such as diabetes type II, coronary heart disease, cardiac infarction, stroke, and others.

Can obesity in Mangalica pigs be explained by mutations in the melanocortin 3 receptor gene?

Supported by National Science Foundation Grant NSF EPS-1158862Supported by National Science Foundation Grant NSF EPS-1158862

Genomic DNA Purification from porcine whole blood. Whole blood was collected from from unrelated blonde Mangalica pigs within the Auburn University research herd. Genomic DNA (gDNA) was purified from these samples using the DNeasy Blood and Tissue Kit (Qiagen, Alameda, CA) according to manufactures. The gDNA was quantified using a Synergy 4 multi-mode plate reader (U.S. BioTek Inc. Seattle, WA) via the Take3 Spec/Gen5 quantization mode.

Primer Design and Polymerase Chain Reaction. The NCBI-Nucleotide sequence for Sus scrofa melanocortin-3 receptor (accession#: NM_001123137.1) was used to design primers for conventional PCR that amplified a region spanning the complete coding region of the putative sequence (Sense:5 -ATGAATGCTTCGTGCTGC-3 Antisense:5 -GCCTCCTACCCCAGGTTC-3 to ′ ′ ′ ′produce an amplicon of 960 base pairs in length. Primers were synthesized by Integrated DNA Technologies (IDT, San Diego, CA). The samples were amplified using a standard PCR protocol performed in a 50 μl mixture containing 100  ng porcine genomic DNA, 0.25 mM dNTPs, 0.4 μM of each of the primers, 1× pfu Turbo DNA polymerase buffer, 1.5 mM MgCl2, and 2.5 U Taq DNA polymerase (Qiagen, La Jolla, CA, USA) with the following parameters: 3 min at 94 °C for one cycle and 1 min at 94 °C, 1 min at 60°C, and 1 min at 72 °C for 40 cycles followed by a final cycle at 72 °C for 10 min. Subsequently, the 960 bp PCR fragment was visualized after electrophoresis with ethidium bromide using a 2% agarose gel, purified with Qiagen PCR purification kit (Qiagen).

DNA Sequencing and Analysis. The concentrations of the purified DNA from PCR reactions were estimated using a Reverse DNA mass ladder (NEB, Ipswich, MA). Purified PCR products were provided to both the core sequencing lab at Davis Sequencing Inc. (Davis, CA) and the Auburn University Core Genomics Lab at a concentration of 20 ng/l with forward primers at 3 nM. Alignment of sequences from 10 pigs and the putative NCBO MC3R sequence (NM_001123137.1) was performed using the Nucleotide Alignment tool of CLC Sequence Viewer version 6.8.1 (CLCbio, Cambridge, MA). The amino acid sequences coded for alleles identified were determined and aligned using the Translate to Protein and Create Alignment Tools of CLC Sequence Viewer version 6.8.1.

Genomic DNA Purification from porcine whole blood. Whole blood was collected from from unrelated blonde Mangalica pigs within the Auburn University research herd. Genomic DNA (gDNA) was purified from these samples using the DNeasy Blood and Tissue Kit (Qiagen, Alameda, CA) according to manufactures. The gDNA was quantified using a Synergy 4 multi-mode plate reader (U.S. BioTek Inc. Seattle, WA) via the Take3 Spec/Gen5 quantization mode.

Primer Design and Polymerase Chain Reaction. The NCBI-Nucleotide sequence for Sus scrofa melanocortin-3 receptor (accession#: NM_001123137.1) was used to design primers for conventional PCR that amplified a region spanning the complete coding region of the putative sequence (Sense:5 -ATGAATGCTTCGTGCTGC-3 Antisense:5 -GCCTCCTACCCCAGGTTC-3 to ′ ′ ′ ′produce an amplicon of 960 base pairs in length. Primers were synthesized by Integrated DNA Technologies (IDT, San Diego, CA). The samples were amplified using a standard PCR protocol performed in a 50 μl mixture containing 100  ng porcine genomic DNA, 0.25 mM dNTPs, 0.4 μM of each of the primers, 1× pfu Turbo DNA polymerase buffer, 1.5 mM MgCl2, and 2.5 U Taq DNA polymerase (Qiagen, La Jolla, CA, USA) with the following parameters: 3 min at 94 °C for one cycle and 1 min at 94 °C, 1 min at 60°C, and 1 min at 72 °C for 40 cycles followed by a final cycle at 72 °C for 10 min. Subsequently, the 960 bp PCR fragment was visualized after electrophoresis with ethidium bromide using a 2% agarose gel, purified with Qiagen PCR purification kit (Qiagen).

DNA Sequencing and Analysis. The concentrations of the purified DNA from PCR reactions were estimated using a Reverse DNA mass ladder (NEB, Ipswich, MA). Purified PCR products were provided to both the core sequencing lab at Davis Sequencing Inc. (Davis, CA) and the Auburn University Core Genomics Lab at a concentration of 20 ng/l with forward primers at 3 nM. Alignment of sequences from 10 pigs and the putative NCBO MC3R sequence (NM_001123137.1) was performed using the Nucleotide Alignment tool of CLC Sequence Viewer version 6.8.1 (CLCbio, Cambridge, MA). The amino acid sequences coded for alleles identified were determined and aligned using the Translate to Protein and Create Alignment Tools of CLC Sequence Viewer version 6.8.1.

1) We were able to successfully isolate gDNA and use conventional PCR to clone the MC3R gene from Mangalica pigs residing at Auburn University.

2) DNA sequence was successfully obtained from four pigs within the herd based upon the criteria that there must be 100% homology between duplicate samples sent to separate DNA sequencing facilities. Based upon these preliminary data, there is no evidence for mutations in the melanocortin 3 receptor gene within Mangalica pigs residing it Auburn University. These results do not support the hypothesis that the extreme obesity seen in Mangalica pigs could be due to defects in the melanocortin 3 receptor.

1) We were able to successfully isolate gDNA and use conventional PCR to clone the MC3R gene from Mangalica pigs residing at Auburn University.

2) DNA sequence was successfully obtained from four pigs within the herd based upon the criteria that there must be 100% homology between duplicate samples sent to separate DNA sequencing facilities. Based upon these preliminary data, there is no evidence for mutations in the melanocortin 3 receptor gene within Mangalica pigs residing it Auburn University. These results do not support the hypothesis that the extreme obesity seen in Mangalica pigs could be due to defects in the melanocortin 3 receptor.

Hypothesis:If mutations in the MC3R gene explain the extreme obesity of Mangalica pigs, then sequencing the wooly pig MCR3 gene will reveal mutations that would be predicted to significantly change the MC3R protein function.

If mutations in the MC3R gene explain the extreme obesity of Mangalica pigs, then sequencing the wooly pig MCR3 gene will reveal mutations that would be predicted to significantly change the MC3R protein function.

Collect whole blood samples to obtain blood

cells (BC)

Purify genomic DNA from BC for use as PCR template

Purify genomic DNA from BC for use as PCR template

Quantify genomic DNA

Conventional PCRTo amplify MC3R gene

Conventional PCRTo amplify MC3R gene

Conventional PCRSequence PCR

product

Purify PCR productPurify PCR product

1. AU core DNA Lab2. Davis Sequencing Lab

Figure 1. Flow diagram depicting the methodology used to clone and sequence the MC3R gene from the Mangalica pigs used in this study. Figure 1. Flow diagram depicting the methodology used to clone and sequence the MC3R gene from the Mangalica pigs used in this study.

Figure 2. Cloning of the MC3R gene using conventional PCR and quantification of purified amplicons using agorose gel electrophoresis. Panel A depicts representative results of PCR reactions using primers directed against the porcine MC3R gene. The DNA ladder indicates size of DNA fragments. The no gDNA lane indicates the results of a PCR reaction in which no template DNA was provided and thus serves as a negative control. PCR amplicons are shown for PCR reactions using gDNA isolated from the blood of pigs 1203, 0401, 1404, and 9071 respectively. Given the reactions yielded a single product of expected size (.96 bp) while no nabd was present in the no gDNA lane, these results indicate that the MC3R gene was succesfully cloned and it was appropriate to continue to the next step in the procedure. Panel B depicts representative results of agarose gel electrophoresis performed to quantity the purified amplicons before sending them to be sequenced. The ladder is a mass ladder. The intensities of ladder bands are compared to those of PCR amplicons to estimate mass of DNA loaded onto the gel.

Figure 2. Cloning of the MC3R gene using conventional PCR and quantification of purified amplicons using agorose gel electrophoresis. Panel A depicts representative results of PCR reactions using primers directed against the porcine MC3R gene. The DNA ladder indicates size of DNA fragments. The no gDNA lane indicates the results of a PCR reaction in which no template DNA was provided and thus serves as a negative control. PCR amplicons are shown for PCR reactions using gDNA isolated from the blood of pigs 1203, 0401, 1404, and 9071 respectively. Given the reactions yielded a single product of expected size (.96 bp) while no nabd was present in the no gDNA lane, these results indicate that the MC3R gene was succesfully cloned and it was appropriate to continue to the next step in the procedure. Panel B depicts representative results of agarose gel electrophoresis performed to quantity the purified amplicons before sending them to be sequenced. The ladder is a mass ladder. The intensities of ladder bands are compared to those of PCR amplicons to estimate mass of DNA loaded onto the gel.

A. B.

Conclusions:

Figure 3. Sequence alignment of nucleotide sequences obtained from cloned Mangalica MC3R genes of individual pigs. Purified PCR amplicons from each pig were sent to both the Davis Sequencing Inc. Lab and the Auburn University Genomics Lab. Sequences were retrieved from each lab and duplicate sequence data was first compared within pig. Sequence data is only reported for a pig if there was 100% homology in the sequence data derived from both labs. Shown at right is the sequence alignment for MC3R genes cloned from pigs 0401, 1404, 2604, and 3709Y using the wild type pig sequence as a reference (sus scrofa MC3R; accession # NM_001123137) as performed using the CLC Sequence Viewer Software. Sequence homology was 100% suggesting there were no mutations present in the Mangalica MC3R gene.

Figure 3. Sequence alignment of nucleotide sequences obtained from cloned Mangalica MC3R genes of individual pigs. Purified PCR amplicons from each pig were sent to both the Davis Sequencing Inc. Lab and the Auburn University Genomics Lab. Sequences were retrieved from each lab and duplicate sequence data was first compared within pig. Sequence data is only reported for a pig if there was 100% homology in the sequence data derived from both labs. Shown at right is the sequence alignment for MC3R genes cloned from pigs 0401, 1404, 2604, and 3709Y using the wild type pig sequence as a reference (sus scrofa MC3R; accession # NM_001123137) as performed using the CLC Sequence Viewer Software. Sequence homology was 100% suggesting there were no mutations present in the Mangalica MC3R gene.

Figure 3.