TY - JOUR

T1 - Characterizing a lethal CAG-ACE2 transgenic mouse model for SARS-CoV-2 infection using Cas9-enhanced nanopore sequencing

AU - Smirnov, Alexander

AU - Nurislamov, Artem

AU - Koncevaya, Galina

AU - Serova, Irina

AU - Kabirova, Evelyn

AU - Chuyko, Eduard

AU - Maltceva, Ekaterina

AU - Savoskin, Maxim

AU - Zadorozhny, Daniil

AU - Svyatchenko, Victor A.

AU - Protopopova, Elena V.

AU - Taranov, Oleg S.

AU - Legostaev, Stanislav S.

AU - Loktev, Valery B.

AU - Serov, Oleg

AU - Battulin, Nariman

N1 - This work was supported by the Ministry of Education and Science of the Russian Federation, agreement 075-15-2024-539. FWNR-2022-0019

PY - 2024/10

Y1 - 2024/10

N2 - The SARS-CoV-2 pandemic has underscored the necessity for functional transgenic animal models for testing. Mouse lines with overexpression of the human receptor ACE2 serve as the common animal model to study COVID-19 infection. Overexpression of ACE2 under a strong ubiquitous promoter facilitates convenient and sensitive testing of COVID-19 pathology. We performed pronuclear microinjections using a 5 kb CAG-ACE2 linear transgene construct and identified three founder lines with 140, 72, and 73 copies, respectively. Two of these lines were further analyzed for ACE2 expression profiles and sensitivity to SARS-CoV-2 infection. Both lines expressed ACE2 in all organs analyzed. Embryonic fibroblast cell lines derived from transgenic embryos demonstrated severe cytopathic effects following infection, even at low doses of SARS-CoV-2 (0,1–1.0 TCID50). Infected mice from the two lines began to show COVID-19 clinical signs three days post-infection and succumbed between days 4 and 7. Histological examination of lung tissues from terminally ill mice revealed severe pathological alterations. To further characterize the integration site in one of the lines, we applied nanopore sequencing combined with Cas9 enrichment to examine the internal transgene concatemer structure. Oxford Nanopore sequencing (ONT) is becoming the gold standard for transgene insert characterization, but it is relatively inefficient without targeted region enrichment. We digested genomic DNA with Cas9 and gRNA against the ACE2 transgene to create ends suitable for ONT adapter ligation. ONT data analysis revealed that most of the transgene copies were arranged in a head-to-tail configuration, with palindromic junctions being rare. We also detected occasional plasmid backbone fragments within the concatemer, likely co-purified during transgene gel extraction, which is a common occurrence in pronuclear microinjections.

AB - The SARS-CoV-2 pandemic has underscored the necessity for functional transgenic animal models for testing. Mouse lines with overexpression of the human receptor ACE2 serve as the common animal model to study COVID-19 infection. Overexpression of ACE2 under a strong ubiquitous promoter facilitates convenient and sensitive testing of COVID-19 pathology. We performed pronuclear microinjections using a 5 kb CAG-ACE2 linear transgene construct and identified three founder lines with 140, 72, and 73 copies, respectively. Two of these lines were further analyzed for ACE2 expression profiles and sensitivity to SARS-CoV-2 infection. Both lines expressed ACE2 in all organs analyzed. Embryonic fibroblast cell lines derived from transgenic embryos demonstrated severe cytopathic effects following infection, even at low doses of SARS-CoV-2 (0,1–1.0 TCID50). Infected mice from the two lines began to show COVID-19 clinical signs three days post-infection and succumbed between days 4 and 7. Histological examination of lung tissues from terminally ill mice revealed severe pathological alterations. To further characterize the integration site in one of the lines, we applied nanopore sequencing combined with Cas9 enrichment to examine the internal transgene concatemer structure. Oxford Nanopore sequencing (ONT) is becoming the gold standard for transgene insert characterization, but it is relatively inefficient without targeted region enrichment. We digested genomic DNA with Cas9 and gRNA against the ACE2 transgene to create ends suitable for ONT adapter ligation. ONT data analysis revealed that most of the transgene copies were arranged in a head-to-tail configuration, with palindromic junctions being rare. We also detected occasional plasmid backbone fragments within the concatemer, likely co-purified during transgene gel extraction, which is a common occurrence in pronuclear microinjections.

KW - COVID-19

KW - Mouse model

KW - Nanopore sequencing

KW - SARS-CoV-2

KW - Transgenic mice

KW - hACE2

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85204798642&origin=inward&txGid=2459b5bf694b038dc0f9a676f4b526f5

UR - https://www.mendeley.com/catalogue/583ddf4a-46b1-3a0d-b5cd-7865ffee26ef/

U2 - 10.1007/s11248-024-00413-w

DO - 10.1007/s11248-024-00413-w

M3 - Article

C2 - 39320390

VL - 33

SP - 453

EP - 466

JO - Transgenic Research

JF - Transgenic Research

SN - 0962-8819

IS - 5

ER -