1. Investigating the Role of Ube2a in the Differentiation of Mouse Embryonic
Stem Cells
Olivia J. Warner – BIO 265
Abstract
Ube2a may play a role in regulating stem cell differentiation. Because stem cells are able to
regenerate and become nearly any type of cell, the ability to regulate this process would be a
significant medical advance in the treatment of many disease and injuries. In order to measure
its expression through real-time quantitative polymerase chain reaction (Q-PCR), it is necessary
to design an optimum primer that will amplify Ube2a in complimentary DNA (cDNA) and not in
genomic DNA and to determine the most favorable concentration of forward and reverse
primers. This research determines the optimum Ube2a primer and its most favorable
concentration for further Q-PCRanalysis to determine its role in stem cell differentiation.
Introduction
Embryonic Stem Cells
Embryonic stem cells (ES cells) are derived from the inner cell mass of early-stage embryonic
blastocysts. ES cells have the unique properties of being able to self-renew indefinitely and be
pluripotent: able to differentiate into ectoderm, endoderm, or mesoderm. In contrast, adult stem
cells are simply multipotent and capable of producing a limited amount of cell types.
Benefits of Regulating Stem Cell Differentiation
Stem cells could play a significant role in the treatment of injuries and diseases requiring tissue
replacement or regenerative medicine due to their ability to limitlessly self-renew and their
capability of becoming virtually any cell type. Therefore it is important that we fully understand
the differentiation process.
The Potential Role of Ube2a in Regulating Stem Cell Differentiation

2. Ubiquitin is a highly-conserved, small protein, consisting of 76 amino acids, that is present in
almost every cell type. Through the process of ubiquitination, ubiquitin attaches to a protein
causing it to be inactivated and marked for degradation. Ubiquitination requires ubiquitin-
activating (E1), ubiquitin-conjugating (E2), and ubuiquitin-protein ligase (E3) enzymes to form a
polyubiquitin chain that binds to a proteasome for degradation. This process is known as the
proteasomal degradation pathway. Ube2a is an ubiquitin-conjugating (E2) enzyme that is
located on X chromosomes. A mutation of Ube2a that results in a premature stop codon is the
cause of a novel X-linked mental retardation syndrome (Nascimento, Otto, De Brouwer, &
Vianna-Morgante, 2006, p. [549]). Also, the absence of Ube2a in oocytes prevents development
beyond the embryonic 2-cell stage (Roest et al., 2004, p. [5485]). Microarray studies provide
evidence that Ube2a may play a role in regulating stem cell differentiation. Recent research
suggests that ubiquitin and the proteasome control and initiate stem cell transcription (Leung et
al., 2008).
The goal of this project is to design and optimize Ube2a primers for Q-PCRin order to analyze
Ube2a expression in both differentiated and undifferentiated embryonic stem cells.
Materials and Methods
Designing PCR Primers
The genomic structure of the Ube2a gene was determined by searching the Mammalian Gene
Collection website at http://lgsun.grc.nia.nih.gov/geneindex/mm8/bin/giU.cgi (Figure 1). The
Ube2a gene structure was used to design 3 primers located in exons that flank large introns that
would derive an amplified product of 100-200 base pairs (bp) from cDNA but would not amplify
genomic DNA due to the presence of the large intervening intron. Primers were chosen based on
the following criteria: equal GC:AT ratio, lacking nucleotide repeats, and having the first 2 5’
bases be a G or C and the final 2 3’ bases be an A or T (Table 1). The primers that best met this
criteria were verified with the Basic Local Alignment Search Tool on the National Center for
Biotechnology Information website to ensure that their sequences were specific to Ube2a.
Ube2a PCR Primer Tests

3. An initial assessment of primers was conducted by standard PCR. Primers were tested at a
concentration of 1 M with 2.5 mM MgCl, 200 M Deoxyribonucleotide triphosphate (dNTP),
and 0.5 l Taq polymerase. Each primer was tested with 2 templates: with no template control
(nuclease-free water), genomic DNA, and cDNA Clone B0263D06 to Ube2a. Cycling
conditions were 95C for 2 minutes, 95C for 30 seconds and 60C for 1 minute and 72C for 30
seconds (repeated 30 times), 72C for 7 minutes, and held at 4C.
Determination of Optimal Primer Concentration for Q-PCR
The forward and reverse primers for Ube2a primer 2 were analyzed using SYBR green
chemistry in a Q-PCRreaction at each possible combination of the following dilutions: 50nM,
100nM, 300nM, 600nM, and 900nM. Each well contained 30 μl 2X SYBR Green master mix, 1
μl cDNA, and 7.4 μl nuclease-free water. Nuclease-free water was added to each well for a final
volume of 60μl.
Confirmation of Linear Amplification
Q-PCRwas performed on Ube2a primer 2 with a concentration of 900nM forward primer and
600 nM reverse primer and cDNA Clone B0263D06 concentrations of 1/10, 1/100, and 1/1000.
The Q-PCRconditions were as above.
Results
Designing PCR Primers
Three primers were designed for Ube2a (Table 1). Primer sites were chosen based on analysis of
the genomic structure of Ube2a (Figure 1).
Ube2a PCR Primer Tests
An initial assessment of primers was conducted by standard PCR. Ube2a primer 1 showed
amplification of genomic DNA at a size of approximately 500 bp, minimal amplification of
cDNA and no amplification in nuclease-free water (Figure 2). Ube2a primer 2 displayed
amplification of cDNA between 100 and 200 bp and did not amplify in either nuclease-free

4. water or genomic DNA (Figure 2). Ube2a primer 3 did not amplify in cDNA, genomic DNA, or
nuclease-free water (Figure 2).
Determination of Optimal Primer Concentration for Q-PCR
The forward and reverse primers for Ube2a primer 2 were analyzed using SYBR green
chemistry in a Q-PCRreaction. At a concentration of 900 nM forward primer and 600 nM
reverse primer, Ube2a primer 2 had the highest level of amplified product with the lowest
presence of nonspecific products (Figure 3). The large peak at approximately 84°C indicates the
amplification of product, while the absence of additional large peaks indicates a lack of
nonspecific products (Figure 3).
Confirmation of Linear Amplification
Q-PCRwas performed on Ube2a primer 2 with a concentration of 900 nM forward primer and
600 nM reverse primer. Threshold cycle (Ct) values were inversely related to the amount of
starting template (Figure 4).

7. Discussion
Of the 3 Ube2a primers that were designed, primer 2 was optimum due to its amplification of
cDNA and its lack of amplification in both nuclease-free water and genomic DNA (Figure 2).
The amplification of 100 to 200 bp cDNA indicates the accuracy of the primer, as it was
designed to amplify 159 bp starting within exon 3 and ending within exon 5 (Figure 1). Ube2a
primer 2 had an optimal concentration of 900 nM forward primer and 600 nM reverse primer,
because this concentration experienced the highest amount of amplified product with the least
amount of nonspecific products (Figure 3). There is a slight peak to the left of the amplified
product, which may indicate the presence of nonspecific products, but it was the least significant
compared to the other Ube2a primer 2 concentrations (Figure 3). Using the optimal
concentration of Ube2a primer 2, linear amplification was verified at the beginning of the

8. exponential phase of Q-PCR which shows that the Ct value is lower for a sample with higher
initial concentration and is higher for a sample with lower initial concentration (Figure 4). This
linear amplification shows a consistent relationship of input DNA to product (Figure 4).
Conclusion
The optimal Ube2a primer is primer 2 with a concentration of 900 nM forward primer and 600
nM reverse primer, because it amplifies cDNA between 100 and 200 bp without amplifying
genomic DNA or nuclease-free water during conventional PCR and it amplifies cDNA during Q-
PCR without amplifying significant amounts of nonspecific products. This successfully
designed and optimized primer can be used to analyze Ube2a expression in differentiated and
undifferentiated stem cells.
References
NIA mouse gene index, mm8. (n.d.). National institute on aging: Intramural
research program. Retrieved from National Institutes of Health website:
http://lgsun.grc.nia.nih.gov/geneindex/mm8/bin/giU.cgi
Leung, A., Geng, F., Daulney, A., Collins, G., Guzzardo, P., & Tansey, W. P.
(2008, October 15). Transcriptional control and the ubiquitin-proteasome
system. In Ernst Schering Foundation Symposium Proceedings: Vol. 2008/1.
The ubiquitin system in health and disease (pp. 75-97). doi:10.1007/
2789_2008_102
Nascimento, R. M. P., Otto, P. A., de Brouwer, A. P. M., & Vianna-Morgante, A.
M. (2006, September). Ube2a, which encodes a ubiquitin-conjugating enzyme,

9. is mutated in a novel x-linked mental retardation syndrome. The American
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