Investigating the Role of Ube2a in the Differentiation of Mouse Embryonic

Stem CellsOlivia 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-PCR analysis 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

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-PCR in 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

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 95C for 2 minutes, 95C for 30 seconds and 60C for 1 minute and 72C for 30

seconds (repeated 30 times), 72C for 7 minutes, and held at 4C.

 

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-PCR reaction 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-PCR was 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-PCR conditions 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

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-PCR reaction. 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-PCR was 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).

Table 1. Primers for Q-PCR Analysis.

Figure 3. Melting curve (dissociation curve) analysis for optimization of Ube2a primer concentration for SYBR Green Q-PCR.This figure shows the melting curve for Ube2a primer 2 at a concentration of 900 nM forward primer and 600nM reverse primer. The large peak at approximately 84°C indicates amplified product.

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

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).

1/10 l 1/100 l 1/1000 lμ μ μ

cDNA dilution

Figure 4. Verification of quantitative amplification for Ube2a primer 2 at a concentration of 900 nM forward primer and 600 nM reverse primer.Ct values were taken at the beginning of the exponential phase of Q-PCR.

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,

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

     Journal of Human Genetics, 79, 549-555.

Roest, H. P., Baarends, W. M., de Wit, J., van Klaveren, J. W., Wassenaar, E.,

     Hoogerbrugge, J. W., . . . Hoejimakers, J. H. J. (2004, June). The

     ubiquitin-conjugating DNA repair enzyme HR6A Is a maternal factor essential

     for early embryonic development in mice. Molecular and Cellular Biology,

     24(12), 5484-5495. doi:10.1128/MCB.24.12.5485–5495.2004