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• W2018187946 abstract "Octamer-binding transcription factor 4 (Oct4) encodes a POU-domain transcription factor (Scholer et al., 1990Scholer H.R. Ruppert S. Suzuki N. Chowdhury K. Gruss P. Nature. 1990; 344: 435-439Crossref PubMed Scopus (594) Google Scholar). The gene is specifically expressed in embryonic stem (ES) cells but can also be detected in adult stem cells such as bone marrow-derived mesenchymal stem cells (Pochampally et al., 2004Pochampally R.R. Smith J.R. Ylostalo J. Prockop D.J. Blood. 2004; 103: 1647-1652Crossref PubMed Scopus (203) Google Scholar). Expression of Oct4 is downregulated during stem cell differentiation. Oct4 plays a critical role in maintaining pluripotency and self-renewal of ES cells (Niwa et al., 2000Niwa H. Miyazaki J. Smith A.G. Nat. Genet. 2000; 24: 372-376Crossref PubMed Scopus (2896) Google Scholar, Pesce and Scholer, 2001Pesce M. Scholer H.R. Stem Cells. 2001; 19: 271-278Crossref PubMed Scopus (670) Google Scholar), but its utility as a marker of pluripotency has been challenged recently by studies suggesting that it is expressed in a variety of differentiated cells, including peripheral blood mononuclear cells (PBMCs) (Tai et al., 2005Tai M.H. Chang C.C. Kiupel M. Webster J.D. Olson L.K. Trosko J.E. Carcinogenesis. 2005; 26: 495-502Crossref PubMed Scopus (518) Google Scholar, Zangrossi et al., 2007Zangrossi S. Marabese M. Broggini M. Giordano R. D'Erasmo M. Montelatici E. Intini D. Neri A. Pesce M. Rebulla P. Lazzari L. Stem Cells. 2007; 25: 1675-1680Crossref PubMed Scopus (144) Google Scholar). However, detection of Oct4 expression by RT-PCR could be prone to artifacts generated by pseudogene transcripts. We therefore have analyzed the sequences of human Oct4 and its pseudogenes and designed PCR primers that can avoid false positive detection of Oct4 expression. Octamer-binding transcription factor 4 (Oct4) encodes a POU-domain transcription factor (Scholer et al., 1990Scholer H.R. Ruppert S. Suzuki N. Chowdhury K. Gruss P. Nature. 1990; 344: 435-439Crossref PubMed Scopus (594) Google Scholar). The gene is specifically expressed in embryonic stem (ES) cells but can also be detected in adult stem cells such as bone marrow-derived mesenchymal stem cells (Pochampally et al., 2004Pochampally R.R. Smith J.R. Ylostalo J. Prockop D.J. Blood. 2004; 103: 1647-1652Crossref PubMed Scopus (203) Google Scholar). Expression of Oct4 is downregulated during stem cell differentiation. Oct4 plays a critical role in maintaining pluripotency and self-renewal of ES cells (Niwa et al., 2000Niwa H. Miyazaki J. Smith A.G. Nat. Genet. 2000; 24: 372-376Crossref PubMed Scopus (2896) Google Scholar, Pesce and Scholer, 2001Pesce M. Scholer H.R. Stem Cells. 2001; 19: 271-278Crossref PubMed Scopus (670) Google Scholar), but its utility as a marker of pluripotency has been challenged recently by studies suggesting that it is expressed in a variety of differentiated cells, including peripheral blood mononuclear cells (PBMCs) (Tai et al., 2005Tai M.H. Chang C.C. Kiupel M. Webster J.D. Olson L.K. Trosko J.E. Carcinogenesis. 2005; 26: 495-502Crossref PubMed Scopus (518) Google Scholar, Zangrossi et al., 2007Zangrossi S. Marabese M. Broggini M. Giordano R. D'Erasmo M. Montelatici E. Intini D. Neri A. Pesce M. Rebulla P. Lazzari L. Stem Cells. 2007; 25: 1675-1680Crossref PubMed Scopus (144) Google Scholar). However, detection of Oct4 expression by RT-PCR could be prone to artifacts generated by pseudogene transcripts. We therefore have analyzed the sequences of human Oct4 and its pseudogenes and designed PCR primers that can avoid false positive detection of Oct4 expression. Pseudogenes are genomic DNA sequences similar to normal genes and are regarded as defunct relatives of functional genes (Vanin, 1985Vanin E.F. Annu. Rev. Genet. 1985; 19: 253-272Crossref PubMed Scopus (527) Google Scholar). There are two types of pseudogenes: processed and nonprocessed pseudogenes. Processed pseudogenes arise by retrotransposition of mRNA and are recognizable by the absence of introns and 5′ promoter sequences (Pavlicek et al., 2002Pavlicek A. Paces J. Zika R. Hejnar J. Gene. 2002; 300: 189-194Crossref PubMed Scopus (41) Google Scholar). This type of pseudogene causes RT-PCR artifacts if the DNase digestion before cDNA synthesis is incomplete. As processed pseudogenes look similar to the mRNA transcript, DNA contamination of the sample leads to amplification of a fragment with the same size as the targeted gene sequence. Nonprocessed pseudogenes result from duplications within the genome and subsequently acquire mutations, which lead to the loss of functionality. This type of pseudogene also has the potential to generate artifacts in RT-PCR analysis. Takeda et al. first described the existence of Oct4 pseudogenes (Takeda et al., 1992Takeda J. Seino S. Bell G.I. Nucleic Acids Res. 1992; 20: 4613-4620Crossref PubMed Scopus (256) Google Scholar), and transcription of some Oct4 pseudogenes was detected in cancer cell lines as well as cancer tissues (Suo et al., 2005Suo G. Han J. Wang X. Zhang J. Zhao Y. Dai J. Biochem. Biophys. Res. Commun. 2005; 337: 1047-1051Crossref PubMed Scopus (160) Google Scholar). In mouse, a recently published paper indicates that some of the putative ES cell-specific pseudogenes may be functionally important and that a novel mouse Oct4 pseudogene can mediate stem cell regulatory function (Lin et al., 2007Lin H. Shabbir A. Molnar M. Lee T. Biochem. Biophys. Res. Commun. 2007; 355: 111-116Crossref PubMed Scopus (41) Google Scholar). These results show that some of the known Oct4 pseudogenes are transcribed in vivo and therefore could lead to RT-PCR artifacts. Although this information has been reported, it has not been considered seriously in a number of recent publications on adult stem cells and tissue analysis referring to Oct4. For cancer tissue, Suo et al., 2005Suo G. Han J. Wang X. Zhang J. Zhao Y. Dai J. Biochem. Biophys. Res. Commun. 2005; 337: 1047-1051Crossref PubMed Scopus (160) Google Scholar suggested analyzing both Oct4 and all the pseudogenes. However, this approach has limitations because the total number of pseudogenes may not yet be known, and use of pseudogene-specific primers requires considerable effort. We have therefore designed a primer set that has the potential to exclude all pseudogenes known to date and detect only the dominant splice variant of Oct4. To approach this project, we began with an initial alignment of Oct4 with its alternative splice variants and its pseudogenes. This alignment served as a basis for an exact primer design. Initially, we clarified the sequence and organization of the functional human Oct4 gene, to allow comparison to the pseudogenes and alternatively spliced transcripts. We searched the NCBI human EST database and examined the UniGene cluster for Oct4 (NM002701). This yielded 13 mRNA sequences and 129 EST sequences. An additional BLASTn search of the human genome using single exons of Oct4 revealed several other highly similar sequences. All these hits encoded complete or partial Oct4 sequences and could therefore represent either functional members of an Oct4 gene family or pseudogenes. Our primary focus was on the 13 mRNA sequences retrieved by the UniGene research and the sequences resulting from BLASTn search. All sequences including the original Oct4 sequence were compared by using the alignment program McAW. The result of the alignment is depicted schematically in Figure 1. In the upper alignment (Figure 1A), the parental Oct4 mRNA (NM002701) and the genomic locus (NW92307) are shown in comparison to alternatively spliced and other Oct4 transcripts originating from chromosome 6. The lower alignment includes the two predicted mRNAs from chromosome 1 XR019318 and chromosome 12 XR16333 and their corresponding DNA retropseudogene sequences NG005794 and NG005793. Also shown in Figure 1B are the RNA sequences NR002304 and DQ486513 and the genomic sequence NT008046 deriving from chromosome 8. The sequence DQ851566 is an Oct4-like mRNA that was also included. This alignment shows the high homology of all pseudogene sequences compared to the original Oct4 sequence. The fact that so many homologous sequences resemble the original Oct4 transcript makes an RT-PCR primer design difficult, as numerous artifacts could arise during amplification. We therefore used our analysis to design primers that are able to exclude amplification of all unwanted transcripts. We designed two different forward primers (see Table S1). One (Oct4_F_P) carries a polymorphism at the 3′ end, which is unique in Oct4 and can distinguish between the parental transcript and the pseudogenes on chromosomes 1 and 8. The other forward primer (Oct4_F) is located in a region that is not homologous to pseudogene sequences. The reverse primer (Oct4_R) is intron spanning and is designed to avoid amplification of DNA. Table S2B shows alignment of two of these primers, Oct4_F and Oct4_R, with alternatively spliced Oct4 sequences and pseudogenes. We also examined some of the previously published primers in light of our alignment. Table S2A shows that one set of published primers (Tondreau et al., 2005Tondreau T. Meuleman N. Delforge A. Dejeneffe M. Leroy R. Massy M. Mortier C. Bron D. Lagneaux L. Stem Cells. 2005; 23: 1105-1112Crossref PubMed Scopus (388) Google Scholar) located in exons 3 and 5 has significant sequence overlap with alternatively spliced Oct4 sequences and pseudogenes. In fact, on chromosome 6 only sequence S81255 has any mismatch with the forward primer of this set. All other sequences have a full match to either the forward or the reverse primer. Similarly for the pseudogenes, in three of eight cases the reverse primer has only a single mismatch. We also analyzed several other published primers used for Oct4 RT-PCR and found that the majority of them lie in exons 2–5 where the highest homology to alternatively spliced variants and pseudogenes occurs. Figure 2 shows the validation of the new proposed primer sets by RT-PCR analysis. Figures 2A and 2B show the results of a preliminary gradient PCR to determine adequate annealing conditions of the used primer. The malignant pluripotent embryonal carcinoma cell line NTERA-2 was used as template, and both cDNA and DNA were tested. As expected, we obtained Oct4 products using cDNA but no products from DNA. These PCR results confirm that our proposed primer sets can discriminate between the parental Oct4 product and retropseudogenes. The products were then further investigated by sequence analysis. The sequences (see Table S3 and Figure S1) confirmed again that only the normal Oct4 transcript (NM002701) was amplified by RT-PCR. Our proposed primer sets were further tested on mononuclear cells (MNCs) from cord blood and PBMCs. Zangrossi et al., 2007Zangrossi S. Marabese M. Broggini M. Giordano R. D'Erasmo M. Montelatici E. Intini D. Neri A. Pesce M. Rebulla P. Lazzari L. Stem Cells. 2007; 25: 1675-1680Crossref PubMed Scopus (144) Google Scholar showed that RT-PCR amplification of total RNA from MNCs and PBMCs yielded products contributed by genomic DNA contamination and mRNA transcripts encoding Oct4. We obtained the same results using their primers. However, we did not obtain products from MNCs and PBMCs using our proposed primer sets. This comparison therefore provides an illustration of how correct primer design can avoid amplification of unwanted or irrelevant sequences. At this point, expression of Oct4 has been reported in adult stem cells as well as in a variety of differentiated cells (Pochampally et al., 2004Pochampally R.R. Smith J.R. Ylostalo J. Prockop D.J. Blood. 2004; 103: 1647-1652Crossref PubMed Scopus (203) Google Scholar, Tai et al., 2005Tai M.H. Chang C.C. Kiupel M. Webster J.D. Olson L.K. Trosko J.E. Carcinogenesis. 2005; 26: 495-502Crossref PubMed Scopus (518) Google Scholar, Tondreau et al., 2005Tondreau T. Meuleman N. Delforge A. Dejeneffe M. Leroy R. Massy M. Mortier C. Bron D. Lagneaux L. Stem Cells. 2005; 23: 1105-1112Crossref PubMed Scopus (388) Google Scholar, Zangrossi et al., 2007Zangrossi S. Marabese M. Broggini M. Giordano R. D'Erasmo M. Montelatici E. Intini D. Neri A. Pesce M. Rebulla P. Lazzari L. Stem Cells. 2007; 25: 1675-1680Crossref PubMed Scopus (144) Google Scholar). However, our analysis shows that it is possible that the detected Oct4 signal came from alternatively spliced or Oct4 pseudogene transcripts rather than from bona fide Oct4 expression. As shown here, precise design of Oct4-specific primers is a prerequisite for accurate RT-PCR analysis. As many pluripotency-associated genes, such as NANOG, also have several pseudogenes (Booth and Holland, 2004Booth H.A. Holland P.W. Genomics. 2004; 84: 229-238Crossref PubMed Scopus (113) Google Scholar, Robertson et al., 2006Robertson M. Stenhouse F. Colby D. Marland J.R. Nichols J. Tweedie S. Chambers I. Mamm. Genome. 2006; 17: 732-743Crossref PubMed Scopus (17) Google Scholar), a similar analysis would seem to be necessary to avoid false positive results derived from other pseudogene products. In addition, the RT-PCR analysis should be verified by western blot analysis. In fact, the available antibodies could prove very valuable in studying the expression and function of Oct4 in view of the putatively expressed pseudogenes and splicing variants of Oct4. We hope that our findings will help other stem cell researchers to devise appropriate tools for RT-PCR analysis and provide an illustration of how misleading artifacts can be avoided by using a detailed alignment as a starting point for designing appropriate primers. We thank Dr. Colin R. MacKenzie (Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University, Düsseldorf) for critically reading this manuscript. The NTERA-2 cell line used in the experiment here was kindly provided by Dr. Axel Methner (Department of Neurology, Heinrich-Heine-University, Düsseldorf). Download .pdf (.09 MB) Help with pdf files Document S1. Three Tables, Two Figures, and Supplemental Experimental Procedures The EMBL accession numbers for sequences termed 1–12 reported in this paper are AM851115–AM851126. An Argument against a Role for Oct4 in Somatic Stem CellsBerg et al.Cell Stem CellOctober 11, 2007In BriefReports of Oct4 expression in somatic and cancer cells have suggested that Oct4 could regulate self-renewal in somatic stem cells as it does in embryonic stem cells. In this issue of Cell Stem Cell, Lengner et al. (2007) provide compelling evidence that Oct4 is neither expressed in nor required for somatic stem cell function. Full-Text PDF Open ArchiveOct4 and Its Pseudogenes Confuse Stem Cell ResearchLiedtke et al.Cell Stem CellFebruary 07, 2008In Brief(Cell Stem Cell 1, 364–366; October 2007) Full-Text PDF Open Archive" @default.
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