Transcript Document

Research Experience in Molecular Biotechnology & Genomics
Summer 2008
Center for Integrated Animal Genomics
Samir M. A’agha1; Dr. Diane Moody Spurlock1; Dawn Elkins1;Hyelee Park1
1Department
of Animal Science, Iowa State University, Ames, Iowa 50011
Variation Analyses in a Holstein population in three genes:
ATGL, CG1-58 and Perilipin for Lipolysis
•No variation was found in the targeted regions of
perilipin or beta-adrenergic receptor.
Introduction
•During early lactation in dairy cows a negative energy
balance is created. This negative energy balance occurs
when energy used for milk production exceeds the
amount of energy consumed.
•To balance this deficiency, energy substrates stored in
adipose tissue are mobilized.
Objective
Identify DNA sequence variation (SNP, single nucleotide
polymorphisms) for genes that participate in the regulation of
lipolysis. Ultimately, associations between alternate alleles of these
genes and energy balance traits will be evaluated.
•In our study we are looking at four genes, perilipin,
beta-adrenergic receptor 1, CGI-58 and ATGL, which
are related to the mobilization of energy from adipose
tissue. We hypothesize that sequence variation in these
genes is associated with energy balance.
Materials and Methods
• Perilipin that has been phosphorylated acts together
with hormone-sensitive lipase to mobilize
triacylgycerols from adipose tissue.
• ATGL catalyzes the initial step in triglyceride
hydrolysis in adipocyte and non-adipocyte lipid
droplets. ATGL also has acylglycerol transacylase
activity, regulates adiposome size, and may be
involved in the degradation of adiposomes.
• ATGL plays an important role in energy homeostasis
and response to starvation, enhancing hydrolysis of
triglycerides and providing free fatty acids to other
tissues to be oxidized in situations of energy depletion.
• CGI-58 interacts with both perilipin and ATGL,
although its role in regulating lipolysis is not well
defined.
•A SNP resulting in an amino acid change has been
reported in the Ensembl database for ATGL.
However, our primers failed to amplify this
targeted region of ATGL. No other SNP were
identified in ATGL.
•CGI-58 was not successfully amplified by the
primers.
Discussion
• Perilipin modulates adipocyte lipid metabolism.
When unphosphorylated, perilipin coats lipid droplets
and prevents the breakdown of triacylglycerols by
hormone-sensitive lipase.
• Beta-adrenergic receptors are activated when
lipolysis is stimulated. Activation of these receptors
leads to a cascade of events that result in
phosphorylation of hormone sensitive lipase and
perilipin.
Results
Figure 1. Assiting in bleeding a
cow via the jugular vein.
Figure 2. Extracting serum
from blood.
•Nine Holstein cows DNA were used in this study.
•Blood for DNA was collected via jugular vein in two, 10 ml tubes with EDTA.
•Buffy coat was extracted from blood.
•DNA was extracted from buffy coat.
•Working solutions were prepared for use in PCR.
•PCR products were designed.
•PCR was used to amplify a region of the genes of interest.
•Agarose gel electrophoresis was done to evaluate PCR products.
•PCR product was prepared for sequencing.
•DNA was sequenced in the DNA Sequencing Facility of Iowa State University.
The SNP in ATGL described in the Ensembl
database would be of great interest in
investigating the function of ATGL in lipolysis.
Although the PCR primers used amplified a
region of ATGL, the size of the PCR product was
smaller than expected and did not include the
potential SNP. Further work with additional PCR
primers is needed to validate the presence of this
SNP in Holstein cattle.
Figure 6. SNP not being reached by the
amplification of primers during PCR.
References
Primers
ATGL FORWARD
5’- GCA CCC TTC CTT CAA CAT GG- 3’
ATGL REVERSE
5’-ATC CCT GTA GCC CTG TTT GC-3’
Primers for Perilipin and beta andregenic
receptor were provided by Dr. Diane
Spurlock lab. ATGL primers were design by
myself and made by Integrated DNA
Technologies.
•James G. Granneman, Hsiao-Ping H.
Moore, Rachel L. Granneman, Andrew
S. Greenberg, Martin S. Obin, and
Zhengxian Zhu. 2006. Analysis of
Lipolytic Protein Trafficking and
Interactions in Adipocytes.
•D. Elkins, H. Park and D. Spurlock.
Phosphorylation of Perilpin is Associated
with Lipolysis in Holstein Cows. Journal
of Dairy Science Vol. 91 No. 7, 2008. pg
2919
• Selection of dairy cattle has focused largely on milk
production traits for decades. Current trends suggest
fitness traits, including health and reproduction, are
compromised in favor of production traits.
• We are looking for variation genes so that we may
select cows to promote the balance of energy
mobilization for milk production as well as fitness
traits.
Acknowledgements
Figure 3. ATGL PCR bands
in gel.
Figure 4. Perilipin PCR bands
in gel.
Figure 5. Beta andregenic
receptor PCR bands in gel.
I would like to thank the NSF for funding my
research, the REU program and staff, Max
Rothschild for giving me the chance to participate
in this program. My mentor, Dr. Diane Spurlock,
for leading my way and bringing me support in
this research. Also Dawn Elkins, graduate
student, for helping me day by day on this
process, Hyelee Park for always being available
when I needed during my experiments.
Program supported by the National Science Foundation Research Experience for Undergraduates
DBI-0552371