Isolation and Cloning of zic Genes in Chick

Abstract

Deficiencies or mutations of various genes, particularly those of the zic family, lead to a number of birth defects, including anencephaly and spina bifida. Currently, research into the role that zic genes play during embryonic development has primarily been done using Xenopus laevis and mouse; however, the use of chick embryos offers new possibilities and methods of researching zic genes. Since only zic1 has been isolated from chick, it has not been possible to use chick for zic gene research. The objective of my research is to isolate and clone the other zic genes in chick (zic2-4). I am using a phage chick cDNA library containing phages carrying the genes. Phage has been arrayed in liquid culture and screened using PCR. Positive wells are diluted, arrayed, and screened again. This process is then repeated a third time to further reduce the number of phage in the pool. The phage is then plated on solid medium and plaques will be screened using PCR. Positive plaques will be isolated and undergo excision to convert to useable plasmids. Currently, we have isolated liquid pools of zic3-containing phages and have begun isolating zic3-containing phages in solid medium.