GENERATION OF AN IMMUNODEFICIENT IFNAR1 KNOCKOUT MOUSE LINE AND CHALLENGES OF THE TRANSGENESIS PROTOCOL USING THE C57BL/6 STRAIN



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Abstract

The laboratory mouse is one of the most extensively studied and well-characterized model organisms in biomedical research and is widely used in virology. However, due to interspecies differences between mice and humans, certain experiments are often impossible because mice are naturally resistant to many human viruses. One possible solution is the generation of knockout mice with reduced immunity. Such models exhibit increased susceptibility to various human pathogens and enable studies of viruses for which no specific mouse models currently exist.

In this study, we report the generation of a knockout mouse line targeting the Ifnar1 gene using two CRISPR-based genome editing strategies. The first involves simultaneous exon deletion and integration of an exon flanked by loxP sites, while the second employs a two-step process to flank the exon with loxP sites without deletion. Both approaches aim to create conditional knockout models but can also produce constitutive knockouts through exon removal.

Traditionally, embryos for microinjection are obtained from first-generation hybrid females, which simplifies the procedure but requires laborious backcrossing to establish congenic lines. Modern techniques now allow the use of inbred strains for embryo collection and microinjection, significantly reducing the time needed to generate genetically modified mice.

In our work, we used the inbred C57BL/6 strain (SPF-vivarium, IC&G SB RAS, Novosibirsk) for embryo collection and microinjection. However, we encountered two main challenges: a marked decrease in the efficiency of the standard superovulation protocol and poor survival rates of injected embryos after transfer. These issues substantially lowered the overall efficiency of the transgenesis workflow and delayed the production of the desired knockout lines.

The most plausible explanation for these difficulties is genetic drift in the local mouse colony. The most effective solution is to obtain inbred mouse strains from international facilities that maintain strict genetic quality control. Such an approach would ensure genetic stability, improve reproducibility, and enhance the efficiency of genome editing experiments in inbred mouse lines.

About the authors

Natalia Anatolyevna Smetannikova

Federal Service for Surveillance on Consumer Rights Protection and Human Well-Being (FBRI SRC VB "Vector", Rospotrebnadzor)

Email: smetannikova@vector.nsc.ru
Scopus Author ID: 6504237445

PhD, senior researcher in the Laboratory of Genome Editing (part of the Department of Genomic Research).

Russian Federation, FBRI SRC VB "Vector", Rospotrebnadzor, Koltsovo, 630559, Russia.

Guzel Ildarovna Davletshina

Federal Service for Surveillance on Consumer Rights Protection and Human Well-Being (FBRI SRC VB "Vector", Rospotrebnadzor)

Email: davletshina_gi@vector.nsc.ru
Scopus Author ID: 57222272758

researcher in the Laboratory of Genome Editing (part of the Department of Genomic Research).

Russian Federation, FBRI SRC VB "Vector", Rospotrebnadzor, Koltsovo, 630559, Russia.

Alexyy Nikolaevich Korablev

Federal Service for Surveillance on Consumer Rights Protection and Human Well-Being (FBRI SRC VB "Vector", Rospotrebnadzor)

Author for correspondence.
Email: korablevalexeyn@gmail.com
ORCID iD: 0000-0002-0571-6935

Candidate of Biological Sciences, senior researcher, Head of the Laboratory of Genome Editing (part of the Department of Genomic Research)

Russian Federation

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