Zic1 is also down regulated in the RFC knockout mouse as shown by microarray analysis. Intriguingly, overexpression of RFC also rescued the effect of the dominant negative constructs of several downstream genes. One possibility is that the epigenetic regulation promoted by RFC is involved directly in the regulation of a panel of the neural crest GRN genes. Thus RFC might work in parallel with the canonical neural crest GRN, although we can not rule out the possibility that this phenomenon is resulted from the cross-regulation between the neural crest genes. Interestingly, overexpression of hMLL1 promoted the expression of Zic1 and FoxD3, but not Snail2 and Twist1, suggesting differential epigenetic regulations of the neural crest genes. Epigenetic mechanisms have also been implicated in folate deficiency-related neural tube defect mouse model Splotch, which carries a loss of function mutation in Pax3. In cells from caudal neural tubes of Pax3 mutant embryos, the expression of the histone demethylases KDM6B decreases and the cells exhibit increased H3K27 methylation. Interestingly, these phenotypes could also be rescued by addition of exogenous folic acid. Pax3 is a key regulator in neural crest development and the H3K4 level of Pax3 transcript region is also developmentally regulated. However, the expression of Pax3 itself was not sensitive to folate levels in our study. These data suggest that different mechanisms are likely involved in the epigenetic regulation of the neural crest genes. There is increasing evidence that chromatin modification plays important roles in vertebrate neural crest development. For example, the CHD member CHD7, an ATP dependent chromatin remodeller, is shown to be essential for neural crest specification and migration. Recently, the histone demethylase JmjD2A has been shown to be required for neural crest induction, which is recruited to the regulatory regions of neural crest genes and thus poises neural crest differentiation. Supplementary folate has been shown to have relatively specific protective effects on neural tube and neural crest cells, suggesting that these cells might be more sensitively regulated by folate related pathways. In addition to folate, microinjection of SAM or VB12 also affects neural crest development in Xenopus embryos, possibly through promoting methylation reactions and thus epigenetic regulations. These data also manifests the role of nutrients in gene transcriptional regulation through epigenetic modification, highlighting the association of dietary intake with epigenetic modification, as well as diseases. Affinity reagents are molecular recognition elements that specifically bind to their targets with high affinity. Thus, their effectiveness constitutes the first and the most important step in pathogen detection and response. Hybridoma monoclonal antibody generation technology has been the most common ICG-001 method for isolating affinity reagents for more than 30 years. However, hybridoma technology requires significant time, cost, and resources. As a result, the demand for high performance affinity reagents for novel molecular targets outpaces the current technology. Currently, a number of synthetic alternatives to hybridoma technology are under development including mRNA and ribosome display, eukaryotic virus display.