Irwin M H, Moffatt R J, Pinkert C A
Dept. of Comparative Medicine, University of Alabama at Birmingham 35294-0019, USA.
Nat Biotechnol. 1996 Sep;14(9):1146-8. doi: 10.1038/nbt0996-1146.
As an alternative to surgically obtaining samples (e.g., tail or tissue biopsy, toe dock, or blood sampling) from weanling mice to screen for transgene integration or other genetic monitoring procedures, we offer a simpler, nonsurgical method. A small amount of saliva, obtained from weanling mice by oral wash using a plastic pipet tip, contains enough oral epithelial cells and lymphocytes to yield sufficient DNA for nested primer polymerase chain reaction (PCR) analysis. The procedure can be repeated many times with minimal stress to the animal, in contrast to tissue biopsy procedures such as tail cutting. Sample analysis is rapid and straightforward; saliva is applied to sample collection paper and then purified using a solid phase DNA purification system. The paper, containing purified DNA, is added directly to PCR cocktail for the first round of amplification. For weanling mice, in the second round of amplification, a small amount of product from the first round is removed and added to PCR cocktail containing the second set of primers. With adult mice, an adequate volume of saliva may be obtained (dependent upon the sensitivity of the particular reaction) to eliminate the need for second-round amplification with nested primers. This technique is reliable, does not require organic solvents, and is more humane than protocols currently in use. Furthermore, this technique could replace hundreds of thousands of surgical biopsies on rodents annually, which are performed for both transgene determination and genetic monitoring procedures.
作为从断奶小鼠获取样本（如尾巴或组织活检、断趾或采血）以筛选转基因整合或进行其他基因监测程序的手术方法的替代方案，我们提供一种更简单的非手术方法。使用塑料移液管头通过口腔冲洗从断奶小鼠获取的少量唾液，含有足够的口腔上皮细胞和淋巴细胞，可产生足够的DNA用于巢式引物聚合酶链反应（PCR）分析。与诸如剪尾等组织活检程序相比，该程序可以对动物造成最小的压力而重复多次。样本分析快速且直接；将唾液应用于样本收集纸，然后使用固相DNA纯化系统进行纯化。含有纯化DNA的纸张直接添加到PCR反应混合物中进行第一轮扩增。对于断奶小鼠，在第二轮扩增中，从第一轮中取出少量产物并添加到含有第二组引物的PCR反应混合物中。对于成年小鼠，可以获得足够体积的唾液（取决于特定反应的灵敏度），从而无需使用巢式引物进行第二轮扩增。该技术可靠，不需要有机溶剂，并且比目前使用的方案更人道。此外，该技术每年可以取代数十万次对啮齿动物进行的手术活检，这些活检用于转基因测定和基因监测程序。
