Lei Y et al. (2013), Generation of gene disruptions by transcription...

XB-ART-47058

Cell Biosci 2013 May 10;31:21. doi: 10.1186/2045-3701-3-21.

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Generation of gene disruptions by transcription activator-like effector nucleases (TALENs) in Xenopus tropicalis embryos.

Lei Y , Guo X , Deng Y , Chen Y , Zhao H .

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Transcription activator-like effector nucleases (TALENs) are novel engineered DNA nucleases, and have been proven to be effective for gene specific targeting in various species. Recently we reported gene disruptions in Xenopus embryos by using TALENs. Here we summarize the protocol that is used in our studies for gene disruption. This protocol covers selection of TALEN targeting sites, TALEN assembly with a modified Golden Gate method, and injection of TALEN mRNAs into Xenopus tropicalis embryos. We also provide details for detection of somatic and germ line transmitted mutations. And finally, we briefly describe establishment of knockout Xenopus lines. This protocol will facilitate broader applications of TALENs in studies of Xenopus biology.

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Species referenced: Xenopus tropicalis
Genes referenced: ets1 etv2

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	Figure 1. Schematic diagrams of TALEN structure and PCR-based assay for detection of mutagenesis. (A) Schematic drawing of TALEN structure. TALEN architecture was optimized for both zebrafish and Xenopus. Each TALEN monomer consists of a nuclear location signal (NLS), 152 amino acids deletion N-terminal, 63 amino acids from C-terminal, the TALE repeat domains, and modified Fok I nuclease domain ELD/KKR. Each TALE repeat unit consists of 34 amino acids, in which the amino acids at positions 12 and 13 are called ‘repeat-variable di-residues’ (RVDs). The RVD determine binding specificity to DNA bases following the code that NG, NI, HD, and NN respectively recognized thymine, adenine, cytosine and guanine. (B) Schematic diagram illustrating binding of TALENs to their targeted DNA sites. Each monomer of a TALEN pair recognizes and binds DNA via upstream or downstream EBE individually. Two Fok I nuclease domains ELD and KKR dimerize and function as endonuclease, generating DNA double strands break (DSB) at spacer between the two EBE sites. (C) PCR assay determining indel mutations induced by TALENs. The DNA fragment coving two EBE sites were amplified with Primer 1/3, the amplicons were then subcloned into pMD18-T using TA cloning. Primer 1/3 were employed to check insertion of targeted sequence after TA cloning. Primer 2 covered the joint region between upstream EBE and the spacer. When indel mutations were amplified in the spacer region, no amplicons would be generated by Primer 2/3. The corresponding plasmids were then sequenced to verify TALEN-induced mutations. Primer 1/4 may also be introduced to this PCR assay for detection of some mutations close to the downstream EBE site, which could not be captured by Primer 2/3. Not drawn to scale.
	Figure 4. Colony PCR results for evaluation of gene disruption. Agarose gel showing an example of colony PCR to detect mutagenesis in ets1-TALEN injected Xenopus tropicalis embryos. The bands amplified by Primer 1/3 in upper panel (~200 bp) indicate the colonies harbor the TALEN targeted sites. PCR assay was also carried out with Primer 2/3 in lower panel. The colonies that can give upper bands but fail to give lower bands were the positive colonies carrying potential mutants. The colonies giving both upper and lower bands did not carry mutants (arrows indicated). Mutations were further confirmed by sequencing.
	Figure 2. Workflow for establishing gene knockout Xenopus lines by TALENs. After selection of TALENs target site and assembly of TALEN plasmids, mRNAs encoding a pair of TALENs were microinjected into Xenopus embryos at one cell stage. The gene disruption efficiency was examined by PCR-based assay and afterwards sequencing. Some of the injected embryos were raised till sexual maturity. Individual G0 founder frog was mated with WT frog for germ line transmission to get F1 frogs. The F1 frogs were genotyped before metamorphosis by cutting tail and were raised to adulthood for intercross. Theoretically, ~25% offspring from F1 Xenopus will be homozygotes frogs carrying a specific gene mutation.
	Figure 3. Agarose gel image showing PCR amplicons from different steps of Golden Gate assembly. pFUS_A vectors contain 1st-10th RVDs repeats (Lane 1) and pFUS_B5 vectors harbor 11th-15th RVDs repeats (Lane 2) in Golden Gate assembly round 1. The smeared ladder bands starting at ~300 bp with increase of approximate every 100 bp indicated successful RVD assembly and ligation to pFUS_A/B. Unsuccessful insertion led to incorrect size of amplicons (Lane 3). Lane 4 and 5 represent colony PCRs for pCS2-TALEN-ELD/KKR vectors carrying RVDs in Golden Gate assembly round 2. Correct PCR fragments amplified by primer pair of CTalF/CTalR are about 200 bp after assembly of the RVDs repeats from pFUS_A, pFUS_B5, and final half RVD repeat, into the pCS2-TALEN-ELD/KKR vectors.

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