Identification of the full-length β-actin sequence and expression profiles in the tree shrew (Tupaia belangeri)

  • Authors:
    • Yu Zheng
    • Chenxia Yun
    • Qihui Wang
    • Wanli W. Smith
    • Jing Leng
  • View Affiliations

  • Published online on: December 15, 2014     https://doi.org/10.3892/ijmm.2014.2040
  • Pages: 519-524
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Abstract

The tree shrew (Tupaia belangeri) diverges from the primate order (Primates) and is classified as a separate taxonomic group of mammals - Scandentia. It has been suggested that the tree shrew can be used as an animal model for studying human diseases; however, the genomic sequence of the tree shrew is largely unidentified. In the present study, we reported the full-length cDNA sequence of the housekeeping gene, β-actin, in the tree shrew. The amino acid sequence of β-actin in the tree shrew was compared to that of humans and other species; a simple phylogenetic relationship was discovered. Quantitative polymerase chain reaction (qPCR) and western blot analysis further demonstrated that the expression profiles of β-actin, as a general conservative housekeeping gene, in the tree shrew were similar to those in humans, although the expression levels varied among different types of tissue in the tree shrew. Our data provide evidence that the tree shrew has a close phylogenetic association with humans. These findings further enhance the potential that the tree shrew, as a species, may be used as an animal model for studying human disorders.

Introduction

The tree shrew (Tupaia belangeri, family Tupaiidae) is a small mammal native to tropical forests, broadly distributed across Southeast Asia (1). It is classified as Scandentia, a separate taxonomic group of mammals and which probably diverged from the primate order (Primates) approximately 85 million years ago (2,3). It has been suggested that tree shrews can potentially be used as an animal model for the study of some human diseases (2). Over the past decades, tree shrew breeding has been on the increase in laboratories (47) and tree shrews have been used as experimental animal models for the study of diseases of the nervous system, visual system and viral infection diseases, such as the human hepatitis virus, herpes simplex virus and influenza virus (712).

However, the genetic background of the tree shrew is largely unidentified as only few tree shrew genes have been fully sequenced. In the GenBank database, there are less than 200 molecules with coding information (partial or complete). To date, there is no record available for the full-length sequence of the tree shrew β-actin (tsACTB) gene. In addition, few antibodies and detecting kits are available for characterizing the tree shrew genome. Due to the lack of genetic and immune background information, the wide application of the tree shrew as a model for the study of human diseases has been greatly impeded.

Housekeeping genes are a group of typically constitutive genes that are required for the maintenance of basic cellular function. When examining human samples, some housekeeping genes are frequently used as an internal reference, such as β-actin (ACTB), 18S ribosomal RNA (18S rRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat-shock protein-90 (Hsp90), β-tubulin, β-2-microglobulin and others (1315). However, as regards the tree shrew, most of the sequences of these genes remain unrevealed.

The actin family is an essential component of the cytoskeleton involved in a wide range of cellular functions, such as cell migration, division, junction formation, cell shape regulation, vesicle trafficking, transcriptional regulation and chromatin remodeling. The family is composed of six isoforms, four of which are muscle-specific, including one γ-isoform for smooth muscle and three α-isoforms for skeletal, cardiac and smooth muscles, respectively; the other two are ubiquitously expressed in the cytoplasm, including the β- and γ-isoform (1618). All isoforms are remarkably similar to each other, with over 93% identity in the amino acid sequence between them (16). Among them, β-actin is a member of the conserved cytoskeleton structural proteins, which are abundant and widely distributed in all eukaryotic cells and play critical roles in cell migration, cell division, wound healing, embryonic development and the immune response (18,19).

In this study, we reported the full-length cDNA sequence of β-actin in the tree shrew and performed phylogenetic analysis of ACTB by constructing a cladogram, further verifying the tsACTB sequence by measuring its expression profiles with quantitative polymerase chain reaction (qPCR) and western blot analysis. Our data provide novel proteomic information on the genetic background of the tree shrew, and enhance the potential of the future use of the tree shrew as a model for the study of human diseases.

Materials and methods

Ethics statement

All procedures related to the use of animals were reviewed and approved by the Review Committee of Guangxi University of Chinese Medicine, Nanning, China in accordance with the Institutional Animal Care and Use regulations and rules.

Tree shrew sampling

Four tree shrews (subfamily of Tupaia belangeri Chinensis) including one male and three females, purchased from Kunming Medical University, Kunming, China were used in this study. All procedures, including animal care, blood collection and tissue collection protocols were conducted in accordance with the Institutional Animal Care and Use regulations and rules.

RACE and sequencing

The 3′- and 5′-rapid amplification of cDNA ends (RACE) was used to clone the tsACTB gene sequence. In brief, total RNA was isolated from the blood samples using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA). The concentration of the RNA was calculated according to the absorbance at 260 and 280 nm which was measured using a Take3 model plate with an Epoch microplate reader (BioTek, Winooski, VT, USA). Based on the published nucleotide sequences of the partial tree shrew ACTB gene (GenBank accession no. AF110103.1), we designed two gene-specific primers (GSP) for the 5′-RACE and 3′-RACE reaction, respectively (Table I). First-strand cDNA was synthesized using the SMARTer RACE cDNA Amplification kit (Clontech, Mountain View, CA, USA); while the amplifications were performed with LA Taq DNA Polymerase (Takara, Dalian, China). The PCR products was electrophoresed on a 1% agarose gel, and then purified using the Gel Extraction kit (CWBio, Beijing, China). Subsequently, the RACE products were linked to the pMD-18T vector (Takara) in accordance with the established protocols, and the constructed plasmids were then sent for sequencing (Life Technologies, Guangzhou, China).

Table I

Primers used in this study.

Table I

Primers used in this study.

PrimerSequence (5′→3′)Application
tsACTB-GSP3RACE CCTGAAGTACCCCATCGAGCACGG3′-RACE amplification
tsACTB-GSP5RACE ATTTGCGCTCAGGGGGAGCGATG5′-RACE amplification
tsACTB-STDf CTGACCCTGAAGTACCCCATCGAGStandard plasmid construction
tsACTB-STDr CGTACTCCTGCTTGCTGATCCACStandard plasmid construction
tsACTB01 (efficiency, 105%)Forward: CACGGCATTGTCACCAACTRT-qPCR
Reverse: TCAGTCAGCAGCACAGGAT
tsACTB02 (efficiency, 101%)Forward: CCTGTATGCCTCTGGTCGTART-qPCR
Reverse: CCACATAGCACAACTTCTCCTT
tsACTB03 (efficiency, 105%)Forward: CACTCTTCCAGCCATCTTRT-qPCR
Reverse: TGTCCACATCACACTTCAT
tsACTB04 (efficiency, 99%)Forward: AGGAGAAGTTGTGCTATGTRT-qPCR
Reverse: ATGGAGTTGAAGGTGGTT

[i] tsACTB, tree shrew β-actin; GSP, gene-specific primers; RACE, rapid amplification of cDNA ends; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Phylogenetic analysis

Alignment was performed using the Clustalw2 program. The phylogenetic tree was computed and constructed by the neighbor-joining method with 5,000 bootstrap replicates, as previously described (20). Values >50% were indicated. All ACTB sequences used in this study are available in GenBank (National Institutes of Health, Bethesda, MD, USA). Their accession numbers are as follows: NM_001101.3, KC215183, NM_001009945.1, AB188274.1, NM_001033084.1, NM_007393.3, NM_031144.3, NM_001172909.1, NM_001281595.1, NM_173979.3, U39357.1, NM_001081838.1, NM_205518.1, NM_001133354.1, NM_001195845.1, NM_001244575.1, NM_001101683.1, NM_213719.1, NM_001088953.1, AF025305.1 and AF012125.1.

Organ and tissue collection

The animals were anesthetized with an injection of 10% chloral hydrate (0.2 ml/100 g) in the abdominal cavity. Warm physiological saline solution was injected through the abdominal vein to expel blood from the organs. When paled, the organ was isolated, weighed and then cut into small tissue sections. These tissue sections were sorted and kept in RNAstore Reagent for the extraction of total RNA, and in Protease Inhibitor Cocktail Buffer (both from CWBio) for protein extraction processes.

Cell culture and collection

According to the datasheet of anti-ACTB antibody (GTX109639; GeneTex, Irvine, CA, USA), the human cell lines, 293T (GNHu17) and HeLa (TCHu187; both from Cell Bank of Shanghai Institutes for Biological Sciences), were used as positive controls. The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS). The cells were collected following a general protocol. In brief, cells were detached from the vessel with Trypsin-EDTA (Life Technologies, Carlsbad, CA, USA) and then washed with phosphate buffer solution.

Construction of standard plasmid for absolute Qpcr

Primers (Table I) were designed based on the sequence (GenBank accession no. KC215183) and then synthesized (Life Technologies, Guangzhou, China). First-strand cDNA was synthesized using the ProtoScript First Strand cDNA Synthesis kit (New England Biolabs, Beijing, China). The fragment for the standard plasmid was amplified using LA Taq DNA Polymerase (Takara). PCR products were electrophoresed on a 1% agarose gel, and purified using the Gel Extraction kit (CWBio). Subsequently, the purified PCR products were cloned into the pMD-18T Vector (Takara). The constructed plasmid was transformed into DH5α E. coli cells and multiplied through propagation of the DH5α E. coli bacteria, and then extracted and purified from bacteria using the TIANprep Mini Plasmid kit (Tiangen, Beijing, China). The purified plasmid was measured by the absorbance method of 260/280 nm, and diluted to a series of concentration gradients: 1010, 109, 108, 107, 106 and 105 copies.

Reverse transcription and qPCR

The mRNA expression of tsACTB in various types of tissue was detected using the absolute qPCR method following the MIQE guidelines, as previously described (21). In brief, total RNA was isolated using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) from the isolated tissues and measured using the absorbance method of 260/280 nm. The PrimeScript II 1st Strand cDNA Synthesis kit (Takara) was used to synthesize the first-strand cDNA from total RNA. The qPCR reactions were carried out using SYBR Premix Ex Taq II (Takara) in the MasterCycler Ep Realplex4 (Eppendorf, Hamburg, Germany). For each reaction, 2 μl cDNA and a final concentration of 50 nmol/l of each primer were used. The cycling profile consisted of an initial denaturation at 95°C for 5 min followed by 40 cycles of 95°C for 20 sec, 55°C for 30 sec, 72°C for 20 sec, followed by melt curve analysis.

Protein extraction and western blot analysis

Tissue protein extraction was carried out using the Tissue Protein Extraction kit, and cellular protein was extracted using the Mammalian Protein Extraction kit (both from CWBio). The total protein concentration was measured using the Pierce BCA Protein Assay kit (Thermo Scientific, Rockford, IL, USA) with an Epoch microplate reader (BioTek). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis were performed following the standard protocol. The sample was loaded with total protein of 10 μg/lane. The anti-ACTB antibody (GTX109639) and secondary HRP-conjugated IgG antibody (GTX213110-01) (both from GeneTex) were applied. Images of the blots were obtained using the Pierce ECL Plus Western Blotting Substrate (Thermo Scientific) in a 4000MM Pro Image Station (Carestream Health, Inc., Rochester, NY, USA).

Data analysis, software and statistical analysis

The primers used in this study were designed by the Primer3 online program, as previously described (22,23). The Clustalw2 online program (24) was used to align and score gene/protein sequences. BioEdit (25) was used to build the alignment graph. MEGA5 (20) was used to compute and construct the phylogenetic tree. The nucleic acid concentration was measured and calculated using Gen5 software (BioTek). Realplex software (Eppendorf, Hamburg, Germany) was used to analyze the results from qPCR. From the results of western blot analysis, graphs were created and analyzed using Molecular Imaging Software (Carestream Health, Inc.). A statistical comparison was carried out with One-Way Analysis of Variance (ANOVA) using the agricolae package (26) in R (27). Charts were built using ggplot2 (28), scales (29) and gridExtra (30) packages in R (27). Charts and graphs were created using Inkscape (31) and the GNU Image Manipulation Program (GIMP) (32).

Results

Full-length cDNA sequence of tsACTB

We identified full-length cDNA sequences of tsACTB from three individual tree shrew specimens. The three sequences were the same. The full-length amino acid sequence of tsACTB was deduced based on its full-length cDNA sequence. The sequence is a cDNA 1,866 bp in length and encodes a protein with 375 amino acids. This is similar to other animals. We deposited the tsACTB cDNA and uploaded the amino acid sequences into GenBank with the accession number: KC215183. To the best of our knowledge, ours is the first study to identify the full-length sequence of tsACTB.

Phylogenetic analysis

To further examine the association between the tree shrew and other species, we compared the tsACTB with ACTB of other animals which can be found in GenBank. The multiple sequence alignment (MSA) results (Fig. 1) revealed that there was no difference in the ACTB amino acid sequence between the tree shrew and humans. Among the different species examined, the ACTB amino acid sequences were exactly the same or differed only in one or two amino acid sequences. With the full-length cDNA sequence of tsACTB from our study, we were able to construct a phylogenetic tree (Fig. 2) using the genetic database from GenBank. Due to the extremely conserved amino acid sequence of ACTB, we constructed this phylogenetic tree with the open reading frame (ORF) of ACTB.

Expression profile analysis of tsACTB in tree shrew tissue

To further determine the tsACTB expression pattern in the tree shrew, qPCR was used to measure the mRNA expression levels of ACTB in various types of tissue from the tree shrew (Fig. 3). We designed four pairs of primers (Table I) to detect tsACTB expression patterns based on the full-length cDNA sequence. The amplification efficiencies of the four pairs of primers are shown in Table I. These pairs of primers are similar in their amplification efficiencies (99–105%). The levels of ACTB expression calculated by the tsACTB03 primer pair were higher than the other three primer pairs, but were similar to those of the other three primer pairs. The expression levels of tsACTB in various types of tissue were compared by One-Way ANOVA followed by Duncan’s multiple range test, with a significance level of P=0.05. According to this statistical analysis, the expression of ACTB can be grouped into two cross groups, marked as ‘a’ and ‘b’, and the crossed parts are marked as ‘ab’ (Fig. 3). The levels of ACTB in the same group did not differ significantly.

Expression pattern of ACTB in the tree shrew

The total identity of ACTB in the amino acid sequence suggests the possibility of the identification of the tsACTB molecule with anti-ACTB antibody which reacts with human ACTB. To assess the ACTB protein expression, western blot analysis was performed to detect tsACTB using an anti-ACTB antibody for humans. All examined tissues expressed the ACTB protein (Fig. 4). The highest level of tsACTB expression in tree shrew tissue was observed in the spleen, bladder and testicles. The lowest levels of tsACTB expression in tree shrew tissue were observed in the liver and muscle (Fig. 4).

Discussion

In this study, we reported the full-length cDNA sequence of ACTB in the tree shrew and performed evolution and sequence analysis. We then identified the sequence using qPCR and verified the protein expression of tsACTB by western blot analysis. Phylogenetic analysis revealed that the tree shrew is classified as Scandentia between the order of Primates and Insectivora. The tsACTB amino acid sequence was found to be completely equal to the ACTB sequence of several species (Fig. 1), including the human, chimpanzee, macaque, mouse, rat guinea pig, hamster cattle, sheep, horse and jungle fowl species. Due to the fact the molecule of ACTB is highly conserved in all species, we constructed a phylogeny tree with the ORF of the ACTB cDNA sequence instead of its amino acid sequence, even though phylogenetic associations are usually analyzed with the amino acid sequence of the molecule. As shown in the cladogram (Fig. 2), the closest phylogenetic relatives to the tree shrew, not belonging to the order of Primates, are the rabbit (Oryctolagus cuniculus) and horse (Equus caballus), which is consistent with the results of previous studies on the tree shrew MHC gene (33,34). Our results support the current classification of the tree shrew, namely that the tree shrew is a small-sized mammal phylogenetically closer to humans than other small-sized experimental animals, such as the mouse, rat, rabbit and guinea pig, but phylogenetically farther than non-human primates, such as the chimpanzee and monkey.

ACTB, a housekeeping gene in humans, mice, rats and other experimental animals, is widely used as an internal reference in quantitative methods, including qPCR and western blot analysis, which are two popularly methods used for the quantification of the mRNA and protein content, respectively. In this study, tsACTB expression was verified to widely and richly exist in each examined tissue of the tree shrew by both qPCR and western blot analysis, although the expression levels varied among the different types of tissue from the tree shrews. Moreover, tsACTB mRNA and protein expression levels were not completely parallel. For instance, our data indicated that the muscle tissue from the tree shrew had significantly high mRNA levels of tsACTB expression and significantly low protein expression levels (Figs. 3 and 4). We also observed that the levels of tsACTB detected by the tsACTB03 primer pair were higher than those detected by the other three qPCR primer pairs (Fig. 3). A possible reason for this is that the product length of the tsACTB primer pair was only 87 bp, which was shorter than the products of the tsACTB01 (104 bp), tsACTB02 (236 bp), and tsACTB04 (208 bp) primer pairs (data not shown). Another possible reason is the high homology of the six isoforms of the actin family and this primer pair may amplify other non-specific isoforms of the actin family. However, as the majority of the cDNA sequences of the tree shrew, including other isoforms of actin family members, are still unknown, the actual reason remains to be revealed.

One of the most crucial steps in experimental design is selecting an internal control gene to normalize the gene expression data according to the objective of the experiment. Our data may prove to be a helpful reference and may aid in the selection of an internal reference in future studies using tree shrews. If experiments are carried out using samples, the reliability of tsACTB expression before and after treatment must be verified in advance. According to our data, tsACTB is a suitable candidate only for comparing expression levels in different types of tissue (the tissue types verified to express similar levels). The primers for used for qPCR and anti-ACTB antibody (human) were good tools for the detection of the mRNA and protein epxression of ACTB in the tree shrew.

Acknowledgments

The authors would like to thank the Laboratory of Molecular Medicine Teaching (Guangxi Medical University) for assistance with qPCR, and would also like to thank Miss Aolei Su and Yinge Qin (Guangxi University of Chinese Medicine) for provding animal care. This study was supported by the National Natural Science Foundation of China (grant no. 31160190), the Guangxi Department of Science and Technology (grant nos. GKG1140003B51 and GKG13470036) and the Guangxi Department of Education [grant no. GJKY(2003)8].

References

1 

Fuchs E and Corbach-Söhle S: Tree Shrews. The UFAW Handbook on the Care and Management of Laboratory and Other Research Animals. Wiley-Blackwell; Oxford: pp. 262–275. 2010, View Article : Google Scholar

2 

Kumar S and Hedges SB: A molecular timescale for vertebrate evolution. Nature. 392:917–920. 1998. View Article : Google Scholar : PubMed/NCBI

3 

Roberts TE, Lanier HC, Sargis EJ and Olson LE: Molecular phylogeny of treeshrews (Mammalia: Scandentia) and the timescale of diversification in Southeast Asia. Mol Phylogenet Evol. 60:358–372. 2011. View Article : Google Scholar : PubMed/NCBI

4 

Shimada A: Studies on Tupaia glis Diard as an experimental animal: its breeding and growth. Jikken Dobutsu. 22(Suppl): S351–S357. 1973.

5 

Schwaier A: Tupaias (tree shrews) - a new animal model for gallstone research; first observations of gallstones. Res Exp Med (Berl). 176:15–24. 1979. View Article : Google Scholar

6 

MacArthur KL, Wu CH and Wu GY: Animal models for the study of hepatitis C virus infection and replication. World J Gastroenterol. 18:2909–2913. 2012. View Article : Google Scholar : PubMed/NCBI

7 

Yang EB, Cao J, Su JJ and Chow P: The tree shrews: useful animal models for the viral hepatitis and hepatocellular carcinoma. Hepatogastroenterology. 52:613–616. 2005.PubMed/NCBI

8 

Yang ZF, Zhao J, Zhu YT, et al: The tree shrew provides a useful alternative model for the study of influenza H1N1 virus. Virol J. 10:1112013.PubMed/NCBI

9 

Walter E, Keist R, Niederöst B, Pult I and Blum HE: Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo. Hepatology. 24:1–5. 1996.PubMed/NCBI

10 

Yan RQ, Su JJ, Huang DR, Gan YC, Yang C and Huang GH: Human hepatitis B virus and hepatocellular carcinoma. I. Experimental infection of tree shrews with hepatitis B virus. J Cancer Res Clin Oncol. 122:283–288. 1996. View Article : Google Scholar : PubMed/NCBI

11 

Xie ZC, Riezu-Boj JI, Lasarte JJ, et al: Transmission of hepatitis C virus infection to tree shrews. Virology. 244:513–520. 1998. View Article : Google Scholar : PubMed/NCBI

12 

Cao J, Yang EB, Su JJ, Li Y and Chow P: The tree shrews: adjuncts and alternatives to primates as models for biomedical research. J Med Primatol. 32:123–130. 2003. View Article : Google Scholar : PubMed/NCBI

13 

de Kok JB, Roelofs RW, Giesendorf BA, et al: Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab Invest. 85:154–159. 2005. View Article : Google Scholar

14 

Eisenberg E and Levanon EY: Human housekeeping genes are compact. Trends Genet. 19:362–365. 2003. View Article : Google Scholar : PubMed/NCBI

15 

Greer S, Honeywell R, Geletu M, Arulanandam R and Raptis L: Housekeeping genes; expression levels may change with density of cultured cells. J Immunol Methods. 355:76–79. 2010. View Article : Google Scholar : PubMed/NCBI

16 

Perrin BJ and Ervasti JM: The actin gene family: function follows isoform. Cytoskeleton (Hoboken). 67:630–634. 2010. View Article : Google Scholar

17 

Gunning P, Weinberger R and Jeffrey P: Actin and tropomyosin isoforms in morphogenesis. Anat Embryol (Berl). 195:311–315. 1997. View Article : Google Scholar

18 

Bunnell TM, Burbach BJ, Shimizu Y and Ervasti JM: β-Actin specifically controls cell growth, migration, and the G-actin pool. Mol Biol Cell. 22:4047–4058. 2011. View Article : Google Scholar : PubMed/NCBI

19 

Nowak D, Skwarek-Maruszewska A, Zemanek-Zboch M and Malicka-Blaszkiewicz M: Beta-actin in human colon adenocarcinoma cell lines with different metastatic potential. Acta Biochim Pol. 52:461–468. 2005.PubMed/NCBI

20 

Tamura K, Peterson D, Peterson N, Stecher G, Nei M and Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 28:2731–2739. 2011. View Article : Google Scholar : PubMed/NCBI

21 

Bustin SA, Benes V, Garson JA, et al: The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 55:611–622. 2009. View Article : Google Scholar : PubMed/NCBI

22 

Untergasser A, Cutcutache I, Koressaar T, et al: Primer3 - new capabilities and interfaces. Nucleic Acids Res. 40:e1152012. View Article : Google Scholar

23 

Koressaar T and Remm M: Enhancements and modifications of primer design program Primer3. Bioinformatics. 23:1289–1291. 2007. View Article : Google Scholar : PubMed/NCBI

24 

Goujon M, McWilliam H, Li W, et al: A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res. 38:W695–W699. 2010. View Article : Google Scholar : PubMed/NCBI

25 

Hall TA: BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series. 41:95–98. 1999.

26 

Mendiburu FD: agricolae: Statistical Procedures for Agricultural Research. R package version 1.1-4. 2013, Available at: http://CRAN.R-Project.org/package=agricolae.

27 

R Core Team: R: A language and environment for statistical computing. R Foundation for Statistical Computing; Vienna, Austria: 2013

28 

Wickham H: ggplot2: Elegant Graphics for Data Analysis. Springer; New York: 2009, View Article : Google Scholar

29 

Wickham H: scales: Scale functions for graphics. R package version 0.2.3. 2012, Available at: http://CRAN.R-project.org.package=scales.

30 

Auguie B: gridExtra: functions in Grid graphics. R package version 0.9.1. 2012, Available at: http://CRAN.R-project.org/package=gridExtra.

31 

The Inkscape Team: Inkscape: an open source vector graphics e ditor. Version 0.48.4. 2012, Available at: http://www.inkscape.org.

32 

Kimball S and Mattis P: GIMP: GNU Image Manipulation Program. Version 2.8.6. 2013, Available at: http://www.gimp.org.

33 

Flügge P, Fuchs E, Günther E and Walter L: MHC class I genes of the tree shrew Tupaia belangeri. Immunogenetics. 53:984–988. 2002. View Article : Google Scholar : PubMed/NCBI

34 

Zhang XH, Dai ZX, Zhang GH, Han JB and Zheng YT: Molecular characterization, balancing selection, and genomic organization of the tree shrew (Tupaia belangeri) MHC class I gene. Gene. 522:147–155. 2013. View Article : Google Scholar : PubMed/NCBI

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Zheng Y, Yun C, Wang Q, Smith WW and Leng J: Identification of the full-length β-actin sequence and expression profiles in the tree shrew (Tupaia belangeri). Int J Mol Med 35: 519-524, 2015
APA
Zheng, Y., Yun, C., Wang, Q., Smith, W.W., & Leng, J. (2015). Identification of the full-length β-actin sequence and expression profiles in the tree shrew (Tupaia belangeri). International Journal of Molecular Medicine, 35, 519-524. https://doi.org/10.3892/ijmm.2014.2040
MLA
Zheng, Y., Yun, C., Wang, Q., Smith, W. W., Leng, J."Identification of the full-length β-actin sequence and expression profiles in the tree shrew (Tupaia belangeri)". International Journal of Molecular Medicine 35.2 (2015): 519-524.
Chicago
Zheng, Y., Yun, C., Wang, Q., Smith, W. W., Leng, J."Identification of the full-length β-actin sequence and expression profiles in the tree shrew (Tupaia belangeri)". International Journal of Molecular Medicine 35, no. 2 (2015): 519-524. https://doi.org/10.3892/ijmm.2014.2040