Analysis of an approach to oviduct-specific expression of modified chicken lysozyme genes.
The -2.7 kb enhancer (E) element of the chicken lysozyme gene domain appears to govern expression of the gene in macrophages but not in oviduct tubular gland cells, the only other site of lysozyme expression. The ultimate goal of our research was to determine whether lysozyme domain variants could be developed that would mainly be expressed in the oviduct so that transgenic birds could be produced that would deposit exogenous protein in the egg white. Accordingly, precise mutations were made by poxvirus-mediated gene targeting in FEF/PU.1 and CCAAT/enhancer-binding protein (C/EBP) transcription factor binding sites in the -2.7 kb E of cloned copies of a specific lysozyme gene variant that includes a hydrophobic pentapeptide tail encoding sequence inserted immediately prior to the stop codon. This variant contains the entire lysozyme domain and is cloned in a lambda bacteriophage vector (lambdaDIILys-HT); the novel tail sequence enables distinction in cell-based expression systems between transcripts of the variant and those of the endogenous gene. These various lysozyme domain mutants, in bacteriophage vector form, were tested for expression in cultured chicken blastodermal cells cotransfected with plasmids encoding the transcription factors C/EBP and v-Myb. In the absence of these plasmids, barely detectable levels of endogenous lysozyme gene transcription resulted in the blastodermal cells. In the presence of the plasmids, however, transcripts of the endogenous gene could be detected as well as varying levels (as evaluated by quantitative real-time PCR) of transcripts of all of the lysozyme domain mutants. These results are discussed in the context of the known role and occurrence of various transcription factors involved in gene expression in differentiating macrophage cells. The ultimate test of expression of the variants in macrophages vs. oviduct cells will be to use them to produce transgenic birds.
Shimizu
Mamiko
M
Department of Animal and Poultry Science, University of Guelph, ON N1G 2W1, Canada.
Losos
Jan K
JK
Gibbins
Ann M Verrinder
AM
D000818:Animals; D001483:Base Sequence; D001756:Blastoderm / Q000166:cytology; D002645:Chickens / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D005818:Genetic Engineering; D008969:Molecular Sequence Data; D009113:Muramidase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D009928:Organ Specificity; D010057:Oviducts / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D014162:Transfection
D009113:Muramidase