Supplementary MaterialsFigure S1: The entire nucleotide sequence from the because of the great difficulty of traditional targeted mutagenesis. pre-mRNA splicing. Mutation of 1 of three feasible branch factors, the polypyrimidine system, as well as the splice acceptor site all triggered exclusion of exon five from mRNA. Interestingly, these exon-skipping mutations allowed usage of cryptic splice acceptor sites within intron four. These data demonstrate that ZFN-mediated gene editing is a highly effective tool for dissection of pre-mRNA splicing regulatory sequences in their endogenous context. Introduction The use of mutagenesis to reveal gene function is a classic technique in biology. The difficulty of achieving targeted mutagenesis in mammalian cells often has necessitated the use of extra-chromosomal or randomly-integrated reporter constructs as a proxy for endogenous gene function. While reporter-based experiments have contributed immensely to our understanding of the cell, loss of correct gene dosage, regulation, and chromatin structure can misrepresent the biology of the endogenous gene. In the case of RNA splicing, use of reporter genes can be unusually problematic as splicing is influenced or regulated by large-scale processes like chromatin modification [1], [2], [3], [4], [5], transcription [6], [7], , and mRNA export [13], [14]. Furthermore, the large size of mammalian genes often simply precludes the use of TH reporter systems to analyze splicing. Retrospective analysis of splicing in cells with naturally-occurring mutations has been informative but is not compatible with directed experimentation and lacks appropriate isogenic controls [15], [16], [17]. Given the centrality of alternative splicing to metazoan biology and disease, techniques that allow investigation of splicing regulation in its natural context are sorely needed. A ZFN pair creates a targeted double-strand break in chromosomal DNA. When repaired inaccurately by the nonhomologous end joining (NHEJ) DNA repair machinery, PF-4136309 tyrosianse inhibitor gene disruption results [18], [19], [20], [21]. Alternately, the homology-directed DNA repair (HDR) pathway can be manipulated to engineer mutations into endogenous genomic loci [22], [23], [24], [25]. In this application, a plasmid with chromosomal DNA sequence flanking the ZFN cleavage site and containing the desired mutation is co-delivered with the ZFNs. The cell can use this donor molecule as a template for DNA repair, resulting in copying of the mutated region into the chromosome (Figure 1A). Open up in another window Shape 1 Alteration of pre-mRNA splicing in response to mutagenesis of crucial regulatory splicing sequences.A) Diagram of homology-directed restoration in the exon five area. Cleavage at the end of exon five stimulates strand invasion by the resected single-strand DNA. Once base pairing is established between the chromosome and the donor, new DNA synthesis and repair of the break using the newly synthesized DNA results in incorporation of the mutated sequence into the chromosome. Thick black lines, homology between the chromosome (top) and the donor plasmid (bottom); black arrow, strand invasion and new DNA synthesis; grey patch in donor plasmid and new DNA, mutation. Exon five and the donor sequence are drawn to scale. B) Putative splice site sequences found within the 3 end of intron four. Potential lariat branch points, the polypyrimidine tract, and the splice acceptor site in intron four are indicated with PF-4136309 tyrosianse inhibitor black bars. The site of ZFN cleavage (shown in grey) is approximately 90 bp from the end of exon five. C) Mutations introduced into intron four and their effect on splicing. The location of specific base changes made in each isogenic cell line are shown in grey. For the branch point C mutation, the terminal C of the consensus sequence shown in part A was not altered. Arrows link the mutation with the gel lane containing RT-PCR products from a cell clone bearing the corresponding mutation. Lane 1; wild-type CHO-K1 cells; lane 2, branch point A mutation; lane 3, branch point B mutation; lane 4, branch point C mutation; lane 5, mutation of branch point C and the polypyrimidine tract (top); lane 6, mutation of the polypyrimidine tract (bottom); lane 7, splice acceptor mutation; lane PF-4136309 tyrosianse inhibitor 8, no template control. The identity and size of bands excised and confirmed by Sanger sequencing is PF-4136309 tyrosianse inhibitor shown to PF-4136309 tyrosianse inhibitor the right of the gel. High-molecular weight bands present in lanes 4C7 are heteroduplex material formed late in the PCR reaction by annealing of normal and exon five-skipped RT-PCR products. Molecular pounds markers are in foundation pairs. The splicing design for every mutation was assayed between 4 and 12 moments; representative data are demonstrated. D) RT-PCR from the wild-type gene in every eight samples acts as a launching control and it is demonstrated below the RT-PCR. The gene can be haploid in CHO cells and its own mutation by NHEJ can lead to lack of a splice acceptor site leading to missing of exon five [26]. We reasoned that built mutagenesis from the endogenous gene via HDR allows us to dissect the sequences necessary for normal splicing. Right here we demonstrate the electricity of.