Targeted genome modification in mice using zinc-finger nucleases - PubMed (original) (raw)
Targeted genome modification in mice using zinc-finger nucleases
Iara D Carbery et al. Genetics. 2010 Oct.
Abstract
Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to >1000 bp in length, among 20-75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research.
Figures
Figure 1.—
The ZFN targeting mechanism. ZFN pairs bind to the target site, and _Fok_I endonuclease domain dimerizes and makes a double strand break between the binding sites. If a DSB is repaired so that the wild-type sequence is restored, ZFNs can bind and cleave again. Otherwise, nonhomologous end joining (NHEJ) introduces deletions or insertions, which change the spacing between the binding sites so that ZFNs might still bind but dimerization or cleavage cannot occur. Insertions or deletions potentially disrupt the gene function.
Figure 2.—
Identification of genetically engineered Mdr1a founders using the Cel-I mutation detection assay. Cleaved bands indicate a mutation is present at the target site (see
materials and methods
). Bands are marked with respective sizes in base pairs. M, PCR marker. One to 44 pups born from injected eggs. The numbers representing the mutant founder animals are underlined.
Figure 3.—
Large deletions in Mdr1a founders. PCR products were amplified using primers located 800 bp upstream and downstream of the ZFN target site. Bands significantly smaller than the 1.6-kb wild-type band indicate large deletions in the target locus. Four founders that were not identified in Figure 2 are underlined.
Figure 4.—
Mdr1a expression in homozygous knockout animals. (A) A schematic of Mdr1a genomic and mRNA structures around the ZFN target site in exon 7, marked with a solid black rectangle. Exons are represented by open rectangles with respective numbers. The size of each exon in base pairs is labeled directly underneath it. Intron sequences are represented by broken bars with size in base pairs underneath. The position of the 396-bp deletion in founder 23 is labeled above intron 6 and exon 7. RT-F and RT-R are the primers used in RT–PCR, located in exons 5 and 9, respectively. (B) Mdr1a expression in tissues. For RT reactions, 40 ng of total RNA was used as template. Normalization of the input RNA was confirmed by GAPDH amplification with or without reverse transcriptase. M, PCR marker; WT, wild-type mouse; F2, _Mdr1a_−/− mouse; K, kidney; I, large intestine; L, liver. Amplicon sizes are marked on the right. (C) Western blot analysis with large intestine. +, positive control, lysate from the human Mdr1-overexpressing SK-N-FI cells (ATCC, Manassas, VA). S3 (15 μl, 10 μl, and 5 μl loaded in each of the three lanes) and S4 (15 μl loaded), the third and fourth supernatant fractions of large intestine membrane preparations (see
materials and methods
). Actin was used as a loading control. Mdr1a+/+, wild-type intestine; _Mdr1a_−/−, intestine from a homozygous knockout mouse derived from founder 23.
Figure 5.—
Identification and genotype of Jag1 founders. (A) _Jag_1 founders identified using the Cel-I mutation detection assay. M, PCR marker; 1–38, pups born from two injection sessions. The numbers of founders are underlined. The sizes in base pairs of uncut and cut bands are labeled on the right. (B) Genotype of the Jag1 founders. Target site sequences of wild type and founders are aligned. ZFN binding sites are in boldface type. A dash represents a deleted nucleotide. One to 4 bp of microhomology that was likely used by NHEJ is underlined. The frameshift (fs), exon skipping (es), or in-frame amino acid loss (if) resulting from each deletion is indicated to the right of each sequence.
Figure 6.—
Identification and genotype of Notch3 founders. M, PCR marker. (A) The Cel-I mutation detection assay was used to identify founders, whose numbers are underlined. (B) Large deletions were detected in founders 1 and 2. (C) Genotype of the Notch3 founders. ZFN binding sites are in boldface type. A dash represents a deleted nucleotide. One to 4 bp of microhomology that was likely used by NHEJ is underlined. All deletions result in frameshift (fs), which is labeled to the right of each sequence.
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