Germ-line knockout heterokaryons of an essential alpha-tubulin gene enable high-frequency gene replacement and a test of gene transfer from somatic to germ-line nuclei in Tetrahymena thermophila
Germ-line knockout heterokaryons of an essential alpha-tubulin gene enable high-frequency gene replacement and a test of gene transfer from somatic to germ-line nuclei in Tetrahymena thermophila
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DOI：
10.1073/pnas.94.4.1310
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发表时间：
1997-02-18
影响因子：
11.1
通讯作者：
Gorovsky, MA
中科院分区：
文献类型：
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作者：
Hai, B;Gorovsky, MA
嗜热四膜虫单倍体基因组只含有一个α-微管蛋白(ATU)基因。通过生物转化，我们在二倍体胚系微核中破坏了ATU基因的两个拷贝之一。杂合的胚系转化子通过与含有缺陷微核的星形菌株交配而使微核纯合子。这种交配被称为第一轮基因组排除，导致了两个不同交配类型的异核克隆，它们在微核中都剔除了ATU基因的拷贝，但在多拷贝体细胞大核中只剔除了野生型基因。当这些异核体交配时，由于新的体细胞大核不含任何α-微管蛋白基因，因此分离的后代细胞不会生长。然而，当这些接合子被功能标记的ATU基因转化时，在新的大核中的同源位置上获得了含有转化的ATU基因的活性转化子。利用野生型ATU基因，可以高效地挽救杂交后代(每亩DNA有900个转化子)。与以前的大核转化方案不同，这一策略应该允许将非常不利(但可行)的突变引入四膜虫，为必要基因的分子和功能研究提供一个强大的工具。这些被敲除的异核体被用来证明从体细胞大核到生殖系微核的基因转移几乎不发生。
The haploid Tetrahymena thermophila genome contains a single alpha-tubulin (ATU) gene. Using biolistic transformation, we disrupted one of the two copies of the ATU gene in the diploid germ-line micronucleus. The heterozygous germ-line transformants were made homozygous in the micronucleus by mating to a star strain containing a defective micronucleus. This mating, known as round 1 genomic exclusion, resulted in two heterokaryon clones of different mating types which have both copies of the ATU gene knocked out in the micronucleus but only wild-type genes in the polycopy somatic macronucleus. When these heterokaryons were mated, the exconjugant progeny cells did not grow because the new somatic macronuclei do not have any alpha-tubulin genes. However, when these conjugants were transformed with a functional marked ATU gene, viable transformants were obtained that contained the transforming ATU gene at the homologous locus in the new macronucleus. The exconjugant progeny could be rescued at a high efficiency (900 transformants per mu g of DNA) with a wild-type ATU gene. Unlike previous macronuclear transformation protocols, this strategy should allow introduction of highly disadvantageous (but viable) mutations into Tetrahymena, providing a powerful tool for molecular and functional studies of essential genes. These knockout heterokaryons were used to demonstrate that gene transfer from somatic macronuclei to germ-line micronuclei occurs rarely if at all.