Identification of the genomic mutation in Epha4(rb-2J/rb-2J) mice.

The EphA4 receptor tyrosine kinase is involved in numerous cell-signalling activities during embryonic development. EphA4 has the ability to bind to both types of ephrin ligands, the ephrinAs and ephrinBs. The C57BL/6J-Epha4rb-2J/GrsrJ strain, denoted Epha4(rb-2J/rb-2J), is a spontaneous mouse mutant that arose at The Jackson Laboratory. These mutants exhibited a synchronous hind limb locomotion defect or "hopping gait" phenotype, which is also characteristic of EphA4 null mice. Genetic complementation experiments suggested that Epha4(rb-2J) corresponds to an allele of EphA4, but details of the genomic defect in this mouse mutant are currently unavailable. We found a single base-pair deletion in exon 9 resulting in a frame shift mutation that subsequently resulted in a premature stop codon. Analysis of the predicted structure of the truncated protein suggests that both the kinase and sterile α motif (SAM) domains are absent. Definitive determination of genotype is needed for experimental studies of mice carrying the Epha4(rb-2J) allele, and we have also developed a method to ease detection of the mutation through RFLP. Eph-ephrin family members are reportedly expressed as numerous isoforms. Hence, delineation of the specific mutation in EphA4 in this strain is important for further functional studies, such as protein-protein interactions, immunostaining and gene compensatory studies, investigating the mechanism underlying the effects of altered function of Eph family of receptor tyrosine kinases on phenotype.

Highly parallel oligonucleotide purification and functionalization using reversible chemistry.

We have developed a cost-effective, highly parallel method for purification and functionalization of 5'-labeled oligonucleotides. The approach is based on 5'-hexa-His phase tag purification, followed by exchange of the hexa-His tag for a functional group using reversible reaction chemistry. These methods are suitable for large-scale (micromole to millimole) production of oligonucleotides and are amenable to highly parallel processing of many oligonucleotides individually or in high complexity pools. Examples of the preparation of 5'-biotin, 95-mer, oligonucleotide pools of >40K complexity at micromole scale are shown. These pools are prepared in up to ~16% yield and 90-99% purity. Approaches for using this method in other applications are also discussed.