TALEN pair injections result in binding of the RVDs to specific DNA nucleotides and thus dimerization of FokI domains, resulting in double stranded breaks³⁹ which can be repaired through non-homologous end joining (NHEJ). NHEJ often introduces errors that result in insertions or deletions (indels). Indels can be identified by amplifying the region surrounding the TALEN target site and digesting the resulting amplicon with a restriction enzyme that cuts within the TALEN spacer region. Alleles without an indel will digest while alleles containing indels that change the restriction enzyme target sequence will not digest, producing a restriction enzyme resistant band (Figure 2).

TALEN injections can likely result in biallelic gene mutations in A. mexicanus²⁹. Thus, some phenotypes may be assessed in founder fish. For example, we evaluated pigmentation in surface fish injected with TALENs targeting oca2, the gene hypothesized to be responsible for albinism in multiple albino populations of cavefish^1,28. We found albino patches in oca2 TALEN-injected fish not present in uninjected fish²⁹ (Figure 3).

For many experiments, it is desirable to have mutant lines of fish for evaluating phenotypes. Founder fish with transmitted mutant alleles can be identified by genotyping progeny from crosses of founder fish to wild type fish (Figure 4).

Figure and Table Legends:

Figure 1: Needle for injecting mRNA.
Photograph of a micropipette prior to being broken used for injecting TALEN mRNA into single celled embryos. Please click here to view a larger version of this figure.

Figure 2: TALEN efficiency for Oca2.
306 bp PCR products from exon 9 of oca2 in Astyanax mexicanus were examined for loss of the restriction enzyme site when different amounts of TALEN mRNA were injected²⁹. The amplicon from a control embryo was digested while a portion of the amplicon was resistant to restriction digest in the pools of 10 TALEN injected embryos. Restriction enzyme digest resistant bands from embryos injected with TALEN mRNA targeted oca2 have been shown to contain indels²⁹. Note that increasing concentrations of mRNA injected results in increased TALEN efficiency (more undigested DNA). Lanes with "-" are undigested, lanes with "+" are digested with restriction enzyme. Please click here to view a larger version of this figure.

Figure 3: Phenotyping founder fish for changes in pigmentation
A. Control uninjected surface A. mexicanus. B. Patch lacking melanophores in a founder surface fish injected with 400 pg TALEN mRNA targeting oca2 (arrow). Please click here to view a larger version of this figure.

Figure 4: Germline transmission of TALEN induced mutations.
306 bp PCR products from exon 9 of oca2 in A. mexicanus were examined for loss of the restriction enzyme site in pools of 10 F₁ fish from an injected founder fish. The amplicon from a control embryo was digested while a portion of the amplicon was resistant to restriction digest in the pools of 10 F₁ embryos. Restriction enzyme digest resistant bands from oca2 F₁s have been shown to contain indels²⁹. Lanes with "-" are undigested, lanes with "+" are digested with restriction enzyme. Please click here to view a larger version of this figure.

Table 1: Example reaction assembly A and B for a TALEN containing RVDs NH-NH-NG-HD-HD-HD-NG-HD-NG-HD-NG-HD-NH-NI-NG.

Table 2: PCR primers for colony PCR, from³⁴.

Table 3: Master mix for 100 µL (15 µL/reaction) for colony PCR 1.

Table 4: PCR program for colony PCR 1.

Table 5: Protocol for second assembly reactions.

Table 6: PCR program for colony PCR 2.

Table 7: Sample needle pulling program.

Table 8: Sample protocol for gene specific PCR.

Table 9: Sample PCR program for gene specific PCR.