Gene Interference Using Antisense Oligodeoxynucleotides on Whole Chick Embryos: Optimal Ring and Roller-Bottle Culture Technique

Antisense oligodeoxynucleotides are being widely used to interfere with specific gene activities in cell-culture systems (1 ,2 ), and there are possible analytical advantages to partial and timed interference in the whole embryo, for certain genes, as compared with targeted null mutations (“knockouts”—see e.g., ref. 3 ). Despite this, and the logical possibility of controlling rigorously for the sequence specificity of effects observed, use of antisense oligos will continue to attract a healthy level of controversy, because of occasional evidence for nongenetically based sequence specificity of particular cellular effects (4 ), and also the possibility of inadvertently targeting other, unknown genes containing matching base sequences. These matters are further discussed below, and in Subheading 4 . The chick blastoderm consists of two or at most three-cell layers at the time many genes with vital roles in development of the basic body plan are being activated, and only one layer, the epiblast, resembles a true epithelium enough to constitute a potential barrier for oligo access to the other two. In fact, there is evidence that epiblast itself is good at uptake of oligos, and it should be remembered that there is evidence for extensive bulk uptake by “cell drinking” (pinocytosis) by ectoderm and neurepithelium, the equivalent cell layer of the young mammalian embryo. This blastoderm can be kept, with or without incubation, in a protein-free nutrient medium for up to 3 h, and then cultured onward in either of two ways.