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目的探讨tk基因分子突变的类型,对不同剂量硝酸羟胺诱导小鼠淋巴瘤L5178Y3.2.7c-tk+/-细胞tk基因突变的杂合缺失进行分析。方法使用硝酸羟胺50～500mg/L剂量对L5178Y细胞进行梯度染毒,分别测定细胞毒性,细胞接种效率,相对悬浮生长率和突变频率。挑选经硝酸羟胺诱导的tk基因突变子(tk-/-)和自发突变体,提取基因组DNA,经等位基因特异性PCR扩增,杂合性缺失(LOH)分析等技术,分析其杂合性缺失的发生。结果大集落和小集落诱导突变体tk基因LOH发生率分别为62.2%和98.9%。由LOH分析的实验结果显示,诱导突变体的LOH在LC与SC之间差异具有统计学意义(P<0.05)。结论功能性等位基因缺失是HAN诱导tk基因分子突变的重要形式之一,在不同集落类型之间可能具有不同的突变机制。
Objective To investigate the types of mutations in tk gene and analyze the heterozygous deletion of tk gene mutation in mouse lymphoma L5178Y3.2.7c-tk +/- cells induced by different doses of hydroxylamine nitrate. Methods L5178Y cells were exposed to 50-500mg / L hydroxylamine nitrate for gradient exposure. Cytotoxicity, cell inoculation efficiency, relative suspension growth rate and mutation frequency were determined. Tk mutants (tk - / -) and spontaneous mutants induced by hydroxylamine nitrate were selected and genomic DNA was extracted. The allele-specific PCR amplification and heterozygous deletion (LOH) analysis were used to analyze the heterozygosity The occurrence of sexual loss. Results The incidence of LOH in tk gene was 62.2% and 98.9%, respectively. The experimental results of LOH analysis showed that there was a significant difference (P <0.05) in the LOH of the induced mutant between LC and SC. Conclusion The deletion of functional alleles is one of the important forms of molecular mutation of tk induced by HAN. Different mutations may exist between different types of colonies.