Generación de una colonia de ratones transgénicos homocigotas K18-hACE2 para la evaluación de candidatos vacunales y terapéuticos contra el SARS-CoV-2

Autores/as

  • Andrea BERENGENO
  • Valentina MATILLER
  • Pablo U. DIAZ
  • Enrique REBELINDO
  • F. M. RODRIGUEZ
  • Ayelen ANWEG
  • P. SILVESTRINI
  • M. L. CATTANEO
  • M. B. PERALTA
  • L. I. DURANTE
  • Ulises S. NOTARO
  • S. CAINELLI
  • P. TABORDA
  • V. STALDER
  • L. ETCHEVERS
  • Florencia REY
  • Natalia R. SALVETTI
  • Hugo H. ORTEGA

DOI:

https://doi.org/10.14409/favecv.v20iSuppl..11212

Palabras clave:

K18-hACE2, genotipificación, modelo preclínico, COVID-19

Resumen

EL SARS-CoV-2 es el agente etiológico responsable de la enfermedad COVID-19 que inició una pandemia desde finales de 2019. Existen distintos modelos animales para esta enfermedad siendo el modelo de ratón transgénico K18-hACE2, originalmente desarrollado para el estudio del SARS-CoV-1, de gran relevancia en el contexto actual. Considerando que los ratones se comercializan como hemicigotas y dada la ausencia de ratones homocigotas para el transgén K18-hACE2, el objetivo de este trabajo fue generar una colonia de ratones homocigotas K18-hACE2-Tg/Tg bajo condiciones ambientales controladas. Para la F1 se utilizaron 10 ratones hemicigotas (7 hembras y 3 machos), de 6-8 semanas de edad, provenientes de The Jackson Laboratory (USA). De la F1 se identificaron mediante genotipificación por PCR 78 animales: 62 hemicigotas (Tg/0) y 16 salvajes (0/0). Aquellos animales identificados como transgénicos se cruzaron con ratones C57BL/6J para seleccionar los parentales homocigotas que generaron 100% de hemicigotas en la F2. De 224 crías analizadas, se obtuvieron 7 parentales homocigotas K18-hACE2-Tg/Tg que fueron utilizados para la colonia fundación de ratones homocigotas K18-hACE2-Tg/Tg. El modelo de ratón homocigota K18-hACE2-Tg/Tg desarrollado en este trabajo podrá ser empleado para el estudio de la patogénesis de la enfermedad y la evaluación de posibles terapéuticos contra el SARS-CoV-2

Citas

Bao L, Deng W, Huang B, Gao H, Liu J, Ren L, Wei Q, Yu P, Xu Y, Qi F, Qu Y, Li F, L Q, Wang W, Xue J, Gong S, Liu M, Wang G, Wang S, Qin C. 2020. The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice. Nature 583: 830-833.

Carossino M, Montanaro P, O´Connell A, Kenney D, Gertje H, Grosz KA, Kurnick SA, Bosmann M, Saeed M, Balasuriya UB, Douam F,Crossland NA. 2021. Fatal neuroinvasion of SARS-CoV-2 in K18-hACE2 mice is partially dependent on hACE2 expression. BioRxiv. doi:10.1101/2021.01.13.425144.

Cockrell AS, Leist SR, Douglas MG, Baric RS. 2018. Modeling pathogenesis of emergent and pre-emergent human coronaviruses in mice. Mammalian Genome 29: 367-383.

Cui J, Li F, Shi ZL. 2019. Origin and evolution of pathogenic coronaviruses. Nat. Rev. Microbiol. 17: 181-192.

Gorbalenya AE, Baker SC, Baric RS, de Groot RJ, Drosten C, Gulyaeva AA, Haagmans BL, Lauber C, Leontovich AM, Neuman BW, Penzar D, Perlman S, Poon LLM, Samborskiy DV, Sidorov IA, Sola I, Ziebuhr J. 2020. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat. Microbiol. 5: 536-544.

Hamming I, Timens W, Bulthuis MLC, Lely AT, Navis GJ, van Goor H. 2004. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J. Pathol. 203: 631-637.

Harmer D, Gilbert M, Borman R, Clark KL. 2002. Quantitative mRNA expression profiling of ACE 2, a novel homologue of angiotensin converting enzyme. FEBS Lett. 532: 107-110.

Hong N, Yu W, Xia J, Shen Y, Yap M, Han W. 2020. Evaluation of ocular symptoms and tropism of SARS-CoV-2 in patients confirmed with COVID-19. Acta. Ophthalmol. 98: e649-e655.

Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, Zhang L, Fan G, Xu J, Gu X, Cheng Z, Yu T, Xia J, Wei Y, Wu W, Xie X, Yin W, Li H, Liu M, Cao B. 2020. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395: 497-506.

Hui KPY, Cheung MC, Perera RAPM, Ng KC, Bui CHT, Ho JCW, Ng MMT, Kuok DIT, Shih KC, Tsao SW, Poon LLM, Peiris M, Nicholls JM, Chan MCW. 2020. Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures. Lancet. Resp. Med. 8: 687-695.

Hulswit RJ, de Haan CAM, Bosch B. 2016. Coronavirus Spike Protein and Tropism Changes. In Adv. Virus. Res. Elservier Inc. Pp. 29-57. ISSN 0065-3527.

Jiang RD, Liu MQ, Chen Y, Shan C, Zhou YW, Shen XR, Li Q, Zhang L, Zhu Y, Si HR, Wang Q, Min J, Wang X, Zhang W, Li B, Zhang HJ, Baric RS, Zhou P, Yang XL, Shi ZL. 2020. Pathogenesis of SARS-CoV-2 in Transgenic Mice Expressing Human Angiotensin-Converting Enzyme 2. Cell 182: 50-58.

Jin X, Lian JS, Hu JH, Gao J, Zheng L, Zhang YM, Hao SR, Jia HY, Cai H, Zhang XL, Yu GD, Xu KJ, Wang XY, Gu JQ, Zhang SY, Ye CY, Jin CL, Lu YF, Yu X, Yang, Y. (2020). Epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (COVID-19) with gastrointestinal symptoms. Gut 69: 1002-1009.

Kumari P, Rothan HA, Natekar JP, Stone S, Pathak H, Strate PG, Arora K, Brinton MA, Kumar M. 2021. Neuroinvasion and encephalitis following intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice. Viruses 13: 1-12.

Lee N, Hui D, Wu A, Chan P, Cameron P, Joynt GM, Ahuja A, Yung MY, Leung CB, To KF, Lui SF, Szeto CC, Chung S,

Sung JJY. 2003. A Major Outbreak of Severe Acute Respiratory Syndrome in Hong Kong. New. Engl. J. Med. 348: 1986-1994.

Li F, Li W, Farzan M, Harrison SC. 2005. Structural biology: Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309: 1864-1868.

Li W, Greenough TC, Moore MJ, Vasilieva N, Somasundaran M, Sullivan JL, Farzan M, Choe H. 2004. Efficient Replication of Severe Acute Respiratory Syndrome Coronavirus in Mouse Cells Is Limited by Murine Angiotensin-Converting Enzyme 2. J. Virol. 78: 11429-11433.

Li W, Moore MJ, Vasilieva N, Su J, Wong SK, Berne MA, Somasundaran M, Sullivan JL, Luzuriaga K, Greenough TC, Choe H, Farzan M. 2003. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426: 450-454.

Li W, Wong SK, Li F, Kuhn JH, Huang IC, Choe H, Farzan M. 2006. Animal Origins of the Severe Acute Respiratory Syndrome Coronavirus: Insight from ACE2-S-Protein Interactions. J. Virol. 80: 4211-4219.

Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, Wang W, Song H, Huang B, Zhu N, Bi Y, Ma X, Zhan F, Wang L, Hu T, Zhou H, Hu Z, Zhou W, Zhao L, Tan W. 2020. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395: 565-574.

Mähler M., Berar M, Feinstein R., Gallagher A, Illgen-Wilcke B, Pritchett-Corning K, Raspa M. 2014. FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units. Lab. Anim. 48: 178-192.

Malik YA. 2020. Properties of coronavirus and SARS-CoV-2. Malay. J. Pathol. 42: 3-11.

McCray PB, Pewe L, Wohlford-Lenane C, Hickey M, Manzel L, Shi L, Netland J, Jia HP, Halabi C, Sigmund CD, Meyerholz DK, Kirby P, Look DC, Perlman S. 2007. Lethal Infection of K18- hACE2 Mice Infected with Severe Acute Respiratory Syndrome Coronavirus. J. Virol. 81: 813-821.

Moreau GB, Burgess SL, Sturek JM, Donlan AN, Petri WA, Mann BJ. 2020. Evaluation of K18-hACE2 Mice as a Model of SARS-CoV-2 Infection. Am. J. Trop. Med. Hyg. 103: 1215-1219.

Muñoz-Fontela C, Dowling WE, Funnell SGP, Gsell PS, Riveros-Balta AX, Albrecht RA, Andersen H, Baric RS, Carroll MW, Cavaleri M, Qin C, Crozier I, Dallmeier K, de Waal L, de Wit E, Delang L, Dohm E, Duprex WP, Falzarano D, Barouch DH. 2020. Animal models for COVID-19. Nature 586: 509-515.

Nguyen HL, Lan PD, Thai NQ, Nissley DA, O’Brien EP, Li MS. 2020. Does SARS-CoV-2 bind to human ACE2 more strongly than does SARS-CoV? J. Phys. Chem. B. 124: 7336-7347.

Oladunni FS, Park JG, Pino PA, Gonzalez O, Akhter A, Allué-Guardia A, Olmo-Fontánez A, Gautam S, Garcia-Vilanova A, Ye C, Chiem K, Headley C, Dwivedi V, Parodi LM, Alfson KJ, Staples HM, Schami A, Garcia JI, Whigham A, Torrelles JB. 2020. Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice. Nat. Commun. 11: 1-17.

Organización Mundial de la Salud (OMS). 2021. Orientaciones sobre la bioseguridad en el laboratorio relacionada con la COVID-19. Orientaciones provisionales. https://apps.who.int/iris/handle/10665/339696 [Consulta 29 de julio de 2021].

Prasad A & Prasad M. 2020. Single Virus Targeting Multiple Organs: What We Know and Where We Are Heading? Front. Med. 7: 1-6.

Ren LL, Wang YM, Wu ZQ, Xiang ZC, Guo L, Xu T, Jiang YZ, Xiong Y, Li YJ, Li XW, Li H, Fan GH, Gu XY, Xiao Y, Gao H, Xu JY, Yang F, Wang XM, Wu C, Wang JW. 2020. Identification of a novel coronavirus causing severe pneumonia in human: a descriptive study. Chinese. Med. J-Peking. 133: 1015-1024.

Rosa RB, Dantas WM, do Nascimento JCF, da Silva MV, de Oliveira RN, Pena LJ. 2021. In vitro and in vivo models for studying SARS-CoV-2, the etiological agent responsible for COVID-19 pandemic. Viruses 13: 1-29.

Su S, Wong G, Shi W, Liu J, Lai ACK, Zhou J, Liu W, Bi Y, Gao GF. 2016. Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses. Trends. Microbiol. 24: 490-502.

Truett G, Heeger P, Mynatt R, Truett A, Walker J, Warman M. 2000. Preparation of PCR- Quality Mouse Genomic DNA with Hot Sodium Hydroxide and Tris (HotSHOT). BioTechniques 29: 52-54.

Tseng CTK, Huang C, Newman P, Wang N, Narayanan K, Watts DM, Makino S, Packard MM, Zaki SR, Chan T,

Peters CJ. 2007. Severe Acute Respiratory Syndrome Coronavirus Infection of Mice Transgenic for the Human Angiotensin-Converting Enzyme 2 Virus Receptor. J. Virol. 81: 1162-1173.

Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D. 2020. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 180: 281-292.

Wan Y, Shang J, Graham R, Baric RS, Li F. 2020. Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus. J. Virol. doi:10.1128/JVI.00127-20.

Weiss SR & Leibowitz JL. 2011. Coronavirus pathogenesis. In Advances in Virus Research. Elsevier Inc. Pp.85-164. ISSN 0065-3527.

Winkler ES, Bailey AL, Kafai NM, Nair S, McCune BT, Yu J, Fox JM, Chen RE, Earnest JT, Keeler SP, Ritter JH, Kang LI, Dort S, Robichaud A, Head R, Holtzman MJ, Diamond MS. 2020. SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function. Nat. Immunol. 21: 1327-1335.

Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, Graham BS, McLellan JS. 2020. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367: 1260-1263.

Wu C, Chen X, Cai Y, Xia J, Zhou X., Xu S, Huang H, Zhang L, Zhou X, Du C, Zhang Y, Song J, Wang S, Chao Y, Yang Z, Xu J, Zhou X, Chen D, Xiong W, Song Y. 2020. Risk Factors Associated with Acute Respiratory Distress Syndrome and Death in Patients with Coronavirus Disease 2019 Pneumonia in Wuhan, China. JAMA Intern. Med. 180: 934-943.

Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, Hu Y, Tao ZW, Tian JH, Pei YY, Yuan ML, Zhang YL, Dai FH, Liu Y, Wang QM, Zheng JJ, Xu L, Holmes EC, Zhang YZ. 2020. A new coronavirus associated with human respiratory disease in China. Nature 579: 265-269.

Xia S, Wu M, Chen S, Zhang T, Ye L, Liu J, Li H. 2020. Long Term Culture of Human Kidney Proximal Tubule Epithelial Cells Maintains Lineage Functions and Serves as an Ex vivo Model for Coronavirus Associated Kidney Injury. Virol Sin. 35: 311-320.

Xu X, Chen P, Wang J, Feng J, Zhou H, Li X, Zhong W, Hao P. 2020. Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission. Sci. China. Life. Sci. 63: 457-460.

Yuan L, Tang Q, Cheng T, Xia N. 2020. Animal models for emerging coronavirus: progress and new insights. Emerg. Microbes. Infec. 9: 949-961.

Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus ADME, Fouchier RAM. 2012. Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia. New. Engl. J. Med. 367: 1814-1820.

Zhang H, Penninger JM, Li Y, Zhong N, Slutsky AS. 2020. Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target. Intens. Care. Med. 46: 586-590.

Zheng J, Wong LYR, Li K, Verma AK, Ortiz ME, Wohlford-Lenane C, Leidinger MR, Knudson CM, Meyerholz DK, McCray PB, Perlman S. 2021. COVID-19 treatments and pathogenesis including anosmia in K18-hACE2 mice. Nature 589: 603-607.

Zheng J. 2020. SARS-CoV-2: An emerging coronavirus that causes a global threat. Int. J. Biol. Sci. 16: 1678-1685.

Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, Si HR, Zhu Y, Li B, Huang CL, Chen H D, Chen J, Luo Y, Guo H, Jiang RD, Liu MQ, Chen Y, Shen XR, Wang X, Shi ZL. 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579: 270-273.

Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R, Niu P, Zhan F, Ma X, Wang D, Xu W, Wu G, Gao GF, Ta W. 2020. A Novel Coronavirus from Patients with Pneumonia in China, 2019. New. Engl. J. Med. 382: 727-733.

Zou X, Chen K, Zou J, Han P, Hao J, Han Z. 2020. Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential risk of different human organs vulnerable to 2019-nCoV infection. Front. Med-Prc. 14: 185-192.

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Publicado

03/04/2022

Cómo citar

BERENGENO, A., MATILLER, V., DIAZ, P. U., REBELINDO, E., RODRIGUEZ, F. M., ANWEG, A., SILVESTRINI, P., CATTANEO, M. L., PERALTA, M. B., DURANTE, L. I., NOTARO, U. S., CAINELLI, S., TABORDA, P., STALDER, V., ETCHEVERS, L., REY, F., SALVETTI, N. R., & ORTEGA, H. H. (2022). Generación de una colonia de ratones transgénicos homocigotas K18-hACE2 para la evaluación de candidatos vacunales y terapéuticos contra el SARS-CoV-2. FAVE Sección Ciencias Veterinarias, 20(Suppl.), 31–39. https://doi.org/10.14409/favecv.v20iSuppl.11212

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