Abstract
BackgroundFloxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking.ResultsHere, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specific and efficient second-round targeting ensures the generation of floxed F1 animals in roughly five months for a wide range of sequence lengths (448 bp to 160 kb reported here).ConclusionsFloxed alleles can be reliably obtained in a predictable timeline using the improved method of electroporation of two gRNA/Cas9 ribonucleoprotein particles (RNPs) and two ssODNs.
Highlights
Flanked by loxP (Floxed) alleles are a crucial portion of conditional knockout mouse models
The availability of hundreds of tissue and cell-type specific Cre driver mouse lines and a wealth of historical data on floxed mice created via embryonic stem cell (ESC) technology have solidified the mouse as a key model organism [1,2,3]
GRNAs were designed in introns flanking one or more exons that are critical for gene function or have coding sequences with lengths that are not the multiple of three. Single-stranded oligodeoxyribonucleotide (ssODN) were designed with a loxP site and a BamHI site inserted directly in the gRNA cleavage sites, flanked by 60 bases of homology sequences on each side. gRNA and ssODN sequences of targets are listed (Additional file 1: Table S1). gRNAs were one of the following formats: in vitro transcripts, chemically modified synthetic crRNA/tracrRNAs
Summary
Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. An efficient and reliable strategy to flox genomic regions of any desired size is still lacking. Floxed alleles are an indispensable tool for the temporal and spatial regulation of gene expression in vivo and functional elucidation of sequence features/chromosomal regions identified in genomics studies. The availability of hundreds of tissue and cell-type specific Cre driver mouse lines and a wealth of historical data on floxed mice created via embryonic stem cell (ESC) technology have solidified the mouse as a key model organism [1,2,3]. With the development of programmable nucleases, single cell embryos are directly manipulated to create diverse mouse models, including floxed mice, with high germline transmission rates [4], circumventing the prerequisite for established ES cell lines.
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