Cloning and Analysis of Lamprey Pax6β CNE

An 878 bp fragment containing the CNE from the Japanese lamprey Pax6β locus plus flanking sequence was cloned by PCR amplification using Phusion high fidelity polymerase (NEB). attB4 and attB1r sequences (underlined in the primers below) were attached to the PCR primers for use with the Gateway recombination cloning system (Invitrogen). The amplified fragment was inserted into the Gateway pP4P1r entry vector using BP clonase and the sequence was verified using M13 forward and reverse primers. Primer sequences used for amplification of the lamprey CNE are:
Lamp6β_NRE_FP-B4:
5′-AACGGGGACAACTTTGTATAGAAAAGTTGGGAGATCGTGATGGAGGTGT-3′ and Lamp6β_NRE_RP-B1r:
5′-AACGGGGACTGCTTTTTTGTACAAACTTGACCCCACGTGTACCGTCTAA-3′ Next, the lamprey CNE-containing pP4P1r entry construct was mixed with a pDONR221 construct containing a gata2 minimal promoter-eGFP-polyA cassette, and recombined using LR Clonase into a destination vector with a Gateway R4-R2 cassette flanked by Tol2 recombination sites to produce the LjPax6β-CNE-gata2-eGFP reporter construct. The minimal gata2 promoter-eGFP reporter cassette has been used to report on the tissue-specific expression patterns driven by a wide variety of linked enhancers and does not produce reporter expression without the presence of linked enhancer elements^{57 (link)}.