Abstract
We recently reported that a goat bacterial artificial chromosome (BAC) clone conferred site-independent expression in transgenic mice of the two loci present within its insert, the ubiquitously expressed Cyclin T1 and the mammary specific β-lactalbumin (αlac) genes. To assess if this vector could target mammary-restricted expression of cDNA, the CAT ORF was introduced by homologous recombination in Escherichia coli in place of the αlac transcription unit. The insert of this modified BAC was injected into mice and three transgenic lines were derived. None of these lines expressed the CAT gene suggesting that the use of long genomic inserts is not sufficient to support the expression of intron-less transgenes. The physically linked goat Cyclin T1 locus was found to be active in all three lines. This observation reinforced the hypothesis that the two loci are localised in two separate chromatin domains.
Highlights
The use of long genomic fragments, such as bacterial artificial chromosome (BAC) or yeast artificial chromosome (YAC) inserts, is often associated with an appropriate expression of the gene of interest in transgenics, [7] for recent review
We recently reported that a 160 kb goat BAC insert conferred position independent, copy-number-related, tissue-specific and developmentally regulated expression to the αlac gene in transgenic mice [15] as well as positionindependent, ubiquitous expression of the physically linked Cyclin T1 gene [9]
To assess if this vector could target mammary-specific expression of a cDNA, we inserted the chloramphenicol acetyl-transferase (CAT) gene in place of the αlac transcription unit (TU) by homologous recombination in Escherichia coli. We report that this substitution silenced the modified goat αlac locus in transgenic mice without affecting the expression of the Cyclin T1 gene
Summary
The use of long genomic fragments, such as bacterial artificial chromosome (BAC) or yeast artificial chromosome (YAC) inserts, is often associated with an appropriate expression of the gene of interest in transgenics, [7] for recent review Subtle mutations of these fragments can be obtained by homologous recombination in Escherichia coli for BAC or in Yeast for YAC. We recently reported that a 160 kb goat BAC insert conferred position independent, copy-number-related, tissue-specific and developmentally regulated expression to the αlac gene in transgenic mice [15] as well as positionindependent, ubiquitous expression of the physically linked Cyclin T1 gene [9] To assess if this vector could target mammary-specific expression of a cDNA, we inserted the chloramphenicol acetyl-transferase (CAT) gene in place of the αlac transcription unit (TU) by homologous recombination in Escherichia coli. We report that this substitution silenced the modified goat αlac locus in transgenic mice without affecting the expression of the Cyclin T1 gene
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