糖基因GLT25D2敲除小鼠的制备与基因型鉴定

目的建立GLT25D2基因敲除小鼠模型，为胶原糖基化修饰分子机制研究奠定工作基础。方法采用胚胎干细胞线性打靶技术，获得同源重组的干细胞，繁殖嵌合体，GLT25D2^＋/-小鼠杂交获得GLT25D2^-/-纯合子小鼠。采用PCR技术对小鼠进行基因型鉴定。结果共获得Founder小鼠4只，其中雄性和雌性各2只。经过8个月的配笼繁殖，共获得GLT25D2一小鼠40只；GLT25D2^＋/-小鼠89只；GLT25D2^-/-小鼠34只。不同基因型比例符合孟德尔遗传规律。对不同基因型小鼠合笼配对观察发现，GLT25D2^-/-小鼠生育功能下降，每胎数量平均3～4只，显著少于GLT25D2^＋/-小鼠之间的每胎小鼠数（平均6～8只）。对20，40，以及60d小鼠的体重观察显示，GLT25D2^-/-小鼠体重显著高于GLT25D2^＋/-和GLT25D2^＋/＋型小鼠，但这种体重差异的趋势随小鼠年龄的增加而逐渐减少。结论GLT25D2基因敲除小鼠发育异常，与野生型小鼠相比，体重增加，繁殖功能降低。

Preparation and genotyping identification of glycogene GLT25D2 knockout mice

Objective To create mouse model of glycogene GLT25D2 knockout, for exploring the molecular mechanism of collagen glycosylation. Methods The neomycin cassette was served as the positive selection marker during the embryonic stem cells targeting step, it was removed by expressing Cre recombinase via a plasmid in the targeted ES cells. Partial exons 2 and total exons 3 were removed, and replaced with loxpNeoloxp. The translation was stopped in exons 2 when meeting the stop code, which is added in-framely into exons 2. The PCR technique was used to identify genotype. Results After stem cells targeting, four founder mice were produced （2 female and 2 male）. Total 163 mice were reproduced, including40 GLT25D2-/- mice, 89 GLT25D2＋/- mice, and 34 GLT25D2＋/＋ mice, after eight months breeding. The proportion was consistent with the Mendelian genetic law. But the GLT25D2 -/- mice showed a relatively low reproductive capability. The GLT25D2-/- mice birth rate of each embryo is 3-4, less than those of GLT25D2 ＋ / - mice （each embro 6-8）. More importantly, the average weigh of GLT25D2-/- mouse significantly higher than those of GLT25D2 ＋/- mice, and GLT25D2 ＋/＋ mice, at 20, 40, and 60 days after birth. The variation trend was gradually decreased with the mice growth. Conclusion The GLT25D2 knockout mice showed a relatively low reproductive capability and abnormal development. Compared with the GLT25D2 ＋/＋ mice, the weight of GLT25D2-/- mice was increased.