摘要
随着小鼠及人类基因组测序的完成,人们越来越意识到小鼠基因组对于解析人类基因组功能及生物医学研究的重要性。虽然研究人员积累了众多基因突变品系,但与果蝇等模式生物相比目前小鼠基因功能研究手段还不丰富,有丝分裂重组介导的嵌合体分析技术和平衡染色体技术还刚刚起步。为进一步发展这两种基因功能分析技术,方便利用各种突变深入研究基因作用机理,本文在小鼠中建立了基于17号染色体的有丝分裂重组系统,并对构建全长17号平衡染色体进行了探索。
有丝分裂重组介导的嵌合体分析技术在胚胎致死基因功能研究及体细胞基因功能筛选中有着广泛的应用。现有的有丝分裂重组系统主要是基于Flp/FRT或Cre/loxP定点重组系统。我们利用基因打靶技术在小鼠17号染色体的近着丝粒端插入了FRT和loxP重组序列以及外源抗原等标记基因。通过泛表达的Pgk-Cre和T细胞特异表达的Lck-Cre诱导,在淋巴细胞中观察到了发生有丝分裂重组的细胞,并发现Lck-Cre能稳定诱导有丝分裂重组。Flp诱导也能使该系统发生有丝分裂重组,但效率要低得多。这一有丝分裂重组系统的建立为进一步对17号染色体进行基因诱变筛选和嵌合体分析等研究奠定了基础
含有倒位的平衡染色体能有效抑制染色体同源重组的发生,在突变筛选和品系保存中发挥了至关重要的作用。建立一个全长平衡染色体需要引入多个倒位,能否选择合适的倒位点并插入可诱导定点重组的loxP序列是建立小鼠平衡染色体的关键一步。我们已经成功地将loxP位点及相应的标记基因在不同的ES细胞中插入到17号染色体的4个不同位置,并证明这些倒位点插入不会引起任何小鼠发育异常。我们同时利用含有两个倒位点插入的ES细胞建立了相应的小鼠品系,由此证明了多次电转后ES细胞依然能够保持多潜能特特性。从而为使用Cre/loxP系统在小鼠17号染色体上制造倒位解决了一个重大的技术难题。
The completion of mouse and human genome sequencing has paved the way of functional analysis of mammalian genes genome wide. However, elegant genetic tools such as mitotic recombination mediated mosaic analysis and balancer chromosomes are still limited in mouse. Mitotic recombination mediated mosaic analysis permits a variety of cellular and molecular studies, including lineage analysis and loss-of-function analysis in intact organisms, while balancer chromosomes are genetic reagents that play important roles in mutagenesis screen and stock maintenance. My thesis focused on establishing a mitotic recombination system and exploring new strategies towards a full-length balancer chromosome for mouse chromosome 17.
To generate a mitotic recombination system, I have integrated recombination sites FRT and loxP, and exogenous markers such as hCD2, at the proximal end of chr. 17. Mitotic recombination events were stably observed in lymphocytes with the presence of T cell specific Lck-Cre. Ubiquitously expressed Pgk-Cre or Actin-Flp recombinase could also induce mitotic recombination, though the latter gave a lower efficiency. Thus, this recombination system could be utilized for mosaic analysis and mutational screens of the genes on chr. 17.
Balancer chromosomes carry inversions that effectively suppress recombination between homologous chromosomes. To generate a balancer chromosome covering full-length mouse chr. 17, I have integrated loxP sequences along with marker genes into four selected loci so that three continuous inversions may be induced thereafter with minimal interference of the viability and fertility. As expected, single locus insertion did not cause developmental abnormalities in vivo. Mouse lines carrying two insertions were also generated from ES cell clones experienced three rounds of electroporation, indicating the feasibility of repeated chromosomal manipulations in ES cells. These studies provided a key step toward the development of a full-length mouse balancer chr. 17 with multiple inversions.
引文
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