An efficient i-GONAD method for creating and maintaining lethal mutant mice using an inversion balancer identified from the C3H/HeJJcl strain

Abstract

As the efficiency of the clustered regularly interspaced short palindromic repeats/Cas system is extremely high, creation and maintenance of homozygous lethal mutants are often difficult. Here, we present an efficient in vivo electroporation method called improved genome editing via oviductal nucleic acid delivery (i-GONAD), wherein one of two alleles in the lethal gene was selectively edited in the presence of a non-targeted B6.C3H-In(6)1J inversion identified from the C3H/HeJJcl strain. This method did not require isolation, culture, transfer, or other in vitro handling of mouse embryos. The edited lethal genes were stably maintained in heterozygotes, as recombination is strongly suppressed within this inversion interval. Using this strategy, we successfully generated the first Tprkb null knockout strain with an embryonic lethal mutation and showed that B6.C3H-In(6)1J can efficiently suppress recombination. As B6.C3H-In(6)1J was tagged with a gene encoding the visible coat color marker, Mitf, the Tprkb mutation could be visually recognized. We listed the stock balancer strains currently available as public bioresources to create these lethal gene knockouts. This method will allow for more efficient experiments for further analysis of lethal mutants.

Recent Advances in In Vivo Genome Editing Targeting Mammalian Preimplantation Embryos

“…Notably, the same group [107] recently demonstrated that i-GONAD can be useful to maintain lethal mutant mice using an inversion balancer identified from the C3H/ HeJJcl strain. As a proof-of-principle, they created the Tp53rk binding protein gene (Tprkb) KO strain with an embryonic lethal mutation through i-GONAD in the presence of a non-targeted B6.C3H-In(6)1 J inversion.…”

“…As a proof-of-principle, they created the Tp53rk binding protein gene (Tprkb) KO strain with an embryonic lethal mutation through i-GONAD in the presence of a non-targeted B6.C3H-In(6)1 J inversion. Iwata et al [107] demonstrated that the edited lethal genes were stably maintained in heterozygotes, as recombination is strongly suppressed within this inversion interval. This strategy may facilitate further analysis of lethal mutants.…”

The Past, Present, and Future of Genetically Engineered Mouse Models for Skeletal Biology

“…Following electroporation of the oviduct, the surgical incision is closed, and normal gestation is resumed. This strategy creates heterozygous, homozygous, and hemizygous mutant alleles, including single nucleotide variants (SNVs) [ 130 , 131 , 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 ]. We have used i -GONAD to create several modified alleles and determined that it is ideal for rapidly and efficiently testing VUSs in genes associated with human birth defects, especially those that may lead to embryonic lethality in a heterozygous state.…”

Recent Advances in the Production of Genome-Edited Animals Using <i>i</i>-GONAD, a Novel <i>in vivo</i> Genome Editing System, and Its Possible Use for the Study of Female Reproductive Systems

“…Since the report of GONAD/i-GONAD, many researchers have reproduced this technology using mice, rats, and hamsters, and some modifications have been made to these technologies [12]. For examples, these include production of KO or KI mice using ssODN as a donor [6,7,, large deletion (LD) of a target site in chromosomes [7,22,37], maintenance of lethal mutant mice using an inversion balancer identified from the C3H/HeJJcl strain [38], production of rodents with floxed alleles [20,22,31] or long DNA fragments at a target locus [7], epitope-tagging at a target gene [32] or in situ somatic gene replacement in oviductal epithelium [39]. Furthermore, these techniques have recently been applied to carcinogenic studies, where oviduct-targeted gene delivery of oncogenes can cause the generation of malignant tumors of oviduct origin [40,41].…”