Challenges for the Characterization of Genetically Modified Animals by the qPCR Technique in the Era of Genomic Editing

Authors

  • Ribrio Ivan Tavares Pereira Batista Laboratório de Fisiologia e Controle da Reprodução, Faculdade de Veterinária, Universidade Estadual do Ceará (UECE), Av. Dr. Silas Munguba 1700, Fortaleza, CE, 60740-903, Brazil;Faculdade de Veterinária, Universidade Federal Fluminense (UFF), Rua Vital Brazil Filho, 64, Niterói, RJ, 24230-340, Brazil
  • Dárcio Ítalo Alves Teixeira Laboratório de Fisiologia e Controle da Reprodução, Faculdade de Veterinária, Universidade Estadual do Ceará (UECE), Av. Dr. Silas Munguba 1700, Fortaleza, CE, 60740-903, Brazil
  • Vicente José de Figueirêdo Freitas Laboratório de Fisiologia e Controle da Reprodução, Faculdade de Veterinária, Universidade Estadual do Ceará (UECE), Av. Dr. Silas Munguba 1700, Fortaleza, CE, 60740-903, Brazil
  • Luciana Magalhães Melo Laboratório de Fisiologia e Controle da Reprodução, Faculdade de Veterinária, Universidade Estadual do Ceará (UECE), Av. Dr. Silas Munguba 1700, Fortaleza, CE, 60740-903, Brazil;Molecular Genetics Research Unit, University Center Fametro (Unifametro), Rua Conselheiro Estelita, 500, Fortaleza,CE, 60010-260, Brazil
  • Joanna Maria Gonçalves Souza-Fabjan Faculdade de Veterinária, Universidade Federal Fluminense (UFF), Rua Vital Brazil Filho, 64, Niterói, RJ, 24230-340,Brazil

DOI:

https://doi.org/10.30564/vsr.v3i1.2877

Abstract

Characterization of genetically modified organisms through determination of zygosity and transgene integration concerning both copy number and genome site is important for breeding a transgenic line and the use of these organisms in the purpose for which it was obtained. Southern blot, fluorescence in situ hybridization or mating are demanding and time-consuming techniques traditionally used in the characterization of transgenic organisms and, with the exception of mating, give ambiguous results. With the emergence of the real-time quantitative PCR technology, different applications have been described for the analysis of transgenic organisms by determination of several parameters to transgenic analysis. However, the accuracy in quantitation by this method can be influenced in all steps of analysis. This review focuses on the aspects that influence pre-analytical steps (DNA extraction and DNA quantification methods), quantification strategies and data analysis in quantification of copy number and zygosity in transgenic animals.

Keywords:

Absolute quantification; Copy number; DNA extraction; Relative quantification; Zygosity

References

[1] Batista RITP, Melo CH, Souza-Fabjan JM, Teixeira DI, Melo LM, Freitas VJ. Phenotypic features of first-generation transgenic goats for human granulocyte-colony stimulation factor production in milk. Biotechnol Lett. 2014 36(11): 2155-62. DOI:https://doi.org/10.1007/s10529-014-1588-0

[2] Houdebine LM. Use of transgenic animals to improve human health and animal production. Reprod Domest Anim. 2005 40(4): 269-81. DOI:https://doi.org/10.1111/j.1439-0531.2005.00596.x

[3] Gordon JW, Ruddle FH. Integration and stable germ line transmission of genes injected into mouse pronuclei. Science. 1981, 214(4526): 1244-6. DOI:https://doi.org/10.1126/science.6272397

[4] Capecchi MR. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet. 2005 Jun;6(6):507- 12. DOI:https://doi.org/10.1038/nrg1619

[5] Ishii T. Reproductive medicine involving genome editing: its clinical and social conundrums. In: Precision Medicine for Investigators, Practitioners and Providers, Academic Press, eds. Joel Faintuch, Salomao Faintuch, pp 419-429, 2020.

[6] B. Tinkle, C. Bieberich, and G. Jay, Molecular approaches involved in mammalian gene transfer: Analysis of transgene integration. In: Transgenic Animal Technology: A Laboratory Handbook, Pinkert CA, eds. Academic Press, San Diego, pp 221-234, 1994.

[7] Tesson L, Heslan JM, Ménoret S, Anegon I. Rapid and accurate determination of zygosity in transgenic animals by real-time quantitative PCR. Transgenic Res. 2002, 11(1):43-8. DOI:https://doi.org/10.1023/a:1013928600442

[8] Ballester M, Castelló A, Ibáñez E, Sánchez A, Folch JM. Real-time quantitative PCR-based system for determining transgene copy number in transgenic animals. Biotechniques. 2004, 37(4): 610-3. DOI:https://doi.org/10.2144/04374ST06

[9] Gaj T, Sirk SJ, Shui SL, Liu J. Genome-Editing Technologies: Principles and Applications. Cold Spring Harb Perspect Biol. 2016, 8(12):a023754. DOI:https://doi.org/10.1101/cshperspect.a023754

[10] Guha TK, Wai A, Hausner G. Programmable Genome Editing Tools and their Regulation for Efficient Genome Engineering. Comput Struct Biotechnol J. 2017, 12;15:146-160. DOI:https://doi.org/10.1016/j.csbj.2016.12.006

[11] Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T. Detection and Identification of Genome Editing in Plants: Challenges and Opportunities. Front Plant Sci. 2019, 12;10:236. DOI:https://doi.org/10.3389/fpls.2019.00236

[12] Yuan JS, Burris J, Stewart NR, Mentewab A, Stewart CN Jr. Statistical tools for transgene copy number estimation based on real-time PCR. BMC Bioinformatics. 2007 Nov 1;8 Suppl 7(Suppl 7):S6. DOI: 10.1186/1471-2105-8-S7-S6. PMID: 18047729; PMCID: PMC2099498.

[13] Grosveld F, van Assendelft GB, Greaves DR, Kollias G. Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell. 1987, 51(6):975-85. DOI:https://doi.org/10.1016/0092-8674(87)90584-8

[14] Bieberich C, Scangos G, Tanaka K, Jay G. Regulated expression of a murine class I gene in transgenic mice. Mol Cell Biol. 1986, 6(4):1339-42. DOI:https://doi.org/10.1128/mcb.6.4.1339

[15] Li J, Brunner AM, Meilan R, Strauss SH. Stability of transgenes in trees: expression of two reporter genes in poplar over three field seasons. Tree Physiol. 2009, 29(2): 299-312. DOI:https://doi.org/10.1093/treephys/tpn028

[16] Mahon KA, Overbeek PA, Westphal H. Prenatal lethality in a transgenic mouse line is the result of a chromosomal translocation. Proc Natl Acad Sci U S A. 1988, 85(4):1165-8. DOI:https://doi.org/10.1073/pnas.85.4.1165

[17] Garrick D, Fiering S, Martin DI, Whitelaw E. Repeat-induced gene silencing in mammals. Nat Genet. 1998 Jan;18(1):56-9. DOI:https://doi.org/10.1038/ng0198-56. PMID: 9425901.

[18] Koike S, Taya C, Aoki J, Matsuda Y, Ise I, Takeda H, Matsuzaki T, Amanuma H, Yonekawa H, Nomoto A. Characterization of three different transgenic mouse lines that carry human poliovirus receptor gene--influence of the transgene expression on pathogenesis. Arch Virol. 1994, 139(3-4): 351-63. DOI:https://doi.org/10.1007/BF01310797

[19] Ida-Hosonuma M, Iwasaki T, Taya C, Sato Y, Li J, Nagata N, Yonekawa H, Koike S. Comparison of neuropathogenicity of poliovirus in two transgenic mouse strains expressing human poliovirus receptor with different distribution patterns. J Gen Virol. 2002, 83(Pt 5):1095-1105. DOI:https://doi.org/10.1099/0022-1317-83-5-1095

[20] Deatly AM, Taffs RE, McAuliffe JM, Nawoschik SP, Coleman JW, McMullen G, Weeks-Levy C, Johnson AJ, Racaniello VR. Characterization of mouse lines transgenic with the human poliovirus receptor gene. Microb Pathog. 1998, 25(1):43-54. DOI:https://doi.org/10.1006/mpat.1998.0212

[21] Chandler KJ, Chandler RL, Broeckelmann EM, Hou Y, Southard-Smith EM, Mortlock DP. Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression. Mamm Genome. 2007, 18(10):693-708. DOI:https://doi.org/10.1007/s00335-007-9056-y

[22] Kong Q, Wu M, Huan Y, Zhang L, Liu H, Bou G, Luo Y, Mu Y, Liu Z. Transgene expression is associated with copy number and cytomegalovirus promoter methylation in transgenic pigs. PLoS One. 2009, 18;4(8):e6679. DOI:https://doi.org/10.1371/journal.pone.0006679

[23] Palmiter RD, Wilkie TM, Chen HY, Brinster RL.Transmission distortion and mosaicism in an unusual transgenic mouse pedigree. Cell. 1984, 36(4):869-77. DOI:https://doi.org/10.1016/0092-8674(84)90036-9

[24] Shitara H, Sato A, Hayashi J, Mizushima N, Yonekawa H, Taya C. Simple method of zygosity identification in transgenic mice by real-time quantitative PCR. Transgenic Res. 2004, 13(2):191-4. DOI:https://doi.org/10.1023/b:trag.0000026084.32492.eb

[25] Guénin S, Mauriat M, Pelloux J, Van Wuytswinkel O, Bellini C, Gutierrez L. Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J Exp Bot. 2009, 60(2):487-93. DOI:https://doi.org/10.1093/jxb/ern305

[26] Burkardt HJ. Standardization and quality control of PCR analyses. Clin Chem Lab Med. 2000 Feb;38(2):87-91. DOI:https://doi.org/10.1515/CCLM.2000.014. PMID: 10834394.

[27] Terry CF, Harris N, Parkes HC. Detection of genetically modified crops and their derivatives: critical steps in sample preparation and extraction. J AOAC Int. 2002 May-Jun;85(3):768-74. PMID: 12083273.

[28] Batista RI, Luciano MC, Teixeira DI, Freitas VJ, Melo LM, Andreeva LE, Serova IA, Serov OL. Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real-time PCR. Biotechnol Prog. 2014, 30(6): 1390-400. DOI:https://doi.org/10.1002/btpr.1946

[29] Demeke T, Ratnayaka I, Phan A. Effects of DNA extraction and purification methods on real-time quantitative PCR analysis of Roundup Ready soybean. J AOAC Int. 2009, 92(4): 1136-44. DOI:https://doi.org/10.1093/jaoac/92.4.1136

[30] P. Corbisier, W. Broothaerts, S. Gioria, H. Schimmel, et al., Toward Metrological Traceability for DNA Fragment Ratios in GM Quantification. 1. Effect of DNA Extraction Methods on the Quantitative Determination of Bt176 Corn by Real-Time PCR. Journal of Agricultural and Food Chemistry, vol. 55, no. 9, 3249-3257, 2007.

[31] Sakurai T, Kamiyoshi A, Watanabe S, Sato M, Shindo T. Rapid zygosity determination in mice by SYBR Green real-time genomic PCR of a crude DNA solution. Transgenic Res. 2008, 17(1):149-55. DOI:https://doi.org/10.1007/s11248-007-9134-7

[32] Rossen L, Nørskov P, Holmstrøm K, Rasmussen OF. Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int J Food Microbiol. 1992 Sep;17(1):37-45. DOI:https://doi.org/10.1016/0168-1605(92)90017-w

[33] Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol. 1997 Oct;63(10):3741-51. DOI:https://doi.org/10.1128/AEM.63.10.3741-3751.1997. PMID: 9327537; PMCID: PMC168683.

[34] Burkhart CA, Norris MD, Haber M. A simple method for the isolation of genomic DNA from mouse tail free of real-time PCR inhibitors. J Biochem Biophys Methods. 2002 Jul 31;52(2):145-9. DOI:https://doi.org/10.1016/s0165-022x(02)00052-0. PMID: 12204418.

[35] Demeke T, Jenkins GR. Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Anal Bioanal Chem. 2010 Mar;396(6):1977- 90. DOI:https://doi.org/10.1007/s00216-009-3150-9. Epub 2009 Sep 30. PMID: 19789856.

[36] Shokere LA, Holden MJ, Jenkins GR. Comparison of fluorometric and spectrophotometric DNA quantification for real-time quantitative PCR of degraded DNA. Food Control 2009, 20(4): 391–401. DOI:https://doi.org/10.1016/j.foodcont.2008.07.009

[37] Rye HS, Dabora JM, Quesada MA, Mathies RA, Glazer AN. Fluorometric assay using dimeric dyes for double- and single-stranded DNA and RNA with picogreen sensitivity. Analytical Biochemistry 1993, 208(1): 144–150. DOI:https://doi.org/10.1006/abio.1993.1020

[38] Wong ML, Medrano JF. Real-time PCR for mRNA quantitation. Biotechniques. 2005 Jul;39(1):75-85. DOI:https://doi.org/10.2144/05391RV01. PMID: 16060372

[39] Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004, 26(6):509-15. DOI:https://doi.org/10.1023/b:bile.0000019559.84305.47

[40] K.J. Livak, and T.D. Schmittgen, “Analysis of relative gene expression data using real-time quantitative PCR and the 2ΔΔC(T) method” Methods, vol. 25, no. 4, pp. 402-408, 2001.

[41] C.J. Smith, and A.M. Osborn, “Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology”, FEMS Microbiology Ecology vol. 67, no. 1, pp. 6-20, 2009.

[42] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25(4):402-8. DOI:https://doi.org/10.1006/meth.2001.1262

[43] Bubner B, Baldwin IT. Use of real-time PCR for determining copy number and zygosity in transgenic plants. Plant Cell Rep. 2004, 23(5):263-71. DOI:https://doi.org/10.1007/s00299-004-0859-y

[44] Schmidt M, Parrott W. Quantitative detection of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) by real-time polymerase chain reaction. Plant Cell Rep. 2001, 20(5):422-428. DOI:https://doi.org/10.1007/s002990100326

[45] Joshi M, Keith Pittman H, Haisch C, Verbanac K. Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice. Biotechniques. 2008, 45(3):247-58. DOI:https://doi.org/10.2144/000112913. PMID: 18778249

[46] Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29(9):e45. DOI:https://doi.org/10.1093/nar/29.9.e45

[47] Pfaffl MW. Quantification strategies in real-time PCR. In: A–Z of Quantitative PCR (Bustin SA,eds.). IUL Biotechnology Series, International University Line, La Jolla, CA, 87-120, 2004.

[48] Ji W, Zhou W, Abruzzese R, Guo W, Blake A, Davis S, Davis S, Polejaeva I. A method for determining zygosity of transgenic zebrafish by TaqMan real-time PCR. Anal Biochem. 2005, 344(2):240-6. DOI:https://doi.org/10.1016/j.ab.2005.06.046

[49] Haurogné K, Bach JM, Lieubeau B. Easy and rapid method of zygosity determination in transgenic mice by SYBR Green real-time quantitative PCR with a simple data analysis. Transgenic Res. 2007,16(1):127-31. DOI:https://doi.org/10.1007/s11248-006-9024-4

[50] Bubner B, Gase K, Baldwin IT. Two-fold differences are the detection limit for determining transgene copy numbers in plants by real-time PCR. BMC Biotechnol. 2004 Jul 13;4:14. DOI:https://doi.org/10.1186/1472-6750-4-14

[51] Mason G, Provero P, Vaira AM, Accotto GP. Estimating the number of integrations in transformed plants by quantitative real-time PCR. BMC Biotechnol. 2002, 2:20. DOI:https://doi.org/10.1186/1472-6750-2-20

[52] J Yuan JS, Wang D, Stewart CN Jr. Statistical methods for efficiency adjusted real-time PCR quantification. Biotechnol J. 2008, 3(1):112-23. DOI:https://doi.org/10.1002/biot.200700169

[53] Yuan JS, Reed A, Chen F, Stewart CN Jr. Statistical analysis of real-time PCR data. BMC Bioinformatics. 2006, 22;7:85. DOI:https://doi.org/10.1186/1471-2105-7-85

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