Flp-In™-Jurkat 细胞系

Flp-In Jurkat细胞的操作需要一些技巧，它们对离心操作非常敏感。你可以尝试使用以下方法来复苏细胞：

•将1管细胞解冻至含有5-7 mL新鲜培养基的T25培养瓶中（没有抗生素）。此时请勿通过离心来去除DMSO，它们对于各种操作非常敏感（包括离心）。通常情况下，解冻后会出现大量碎片。
•解冻后24小时之后，以900 rpm 2-3分钟的离心条件沉淀细胞，并使用5 mL新鲜培养基轻柔重悬细胞。将所有细胞整体离心会显著减少培养物中的碎片数量。
•培养5或6天时即可加入筛选剂。在1.5周的时间内，按照2-3天的频率持续传代细胞（离心沉淀细胞），每一次都将细胞重悬至仅含5-7 ml新鲜培养基的T25培养瓶中，以重建细胞密度。Jurkat细胞非常细小和容易聚团，而且扩增得很慢。
•仅在1.5周左右后，可以尝试将细胞传入T75培养瓶中进行扩增。

在理论上来讲，用户能够实现Flp-In表达载体的多重整合效果——这其中包含了一个FRT-特异性的整合事件和一个随机的第二位点整合事件。不过，随机整合的发生概率相对较低。转染过程中所用DNA的有限数量将减少第二位点的整合概率。我们向293细胞（缺少FRT位点）中转染了pcDNA5/FRT载体，并在筛选了200个以上的克隆后，鉴定到一个潜在的第二整合位点。用户可通过Southern杂交来检测DNA的整合位点。单一整合元件会显示为独立的一个条带；两个整合位点：两个条带；三个位点，三条带，如此延续。我们将一些Flp-In表达细胞系培养了四个月以上，无论是否向培养体系中加入潮霉素，均未发现Flp-In表达载体发生任何形式的丢失。

我们提供向HEK293细胞中稳定整合了pFRT/lacZeo和pcDNA6/TR的Flp-In T-REx系统。该细胞系经功能性测试，能够有效调控目的基因的表达。

Flp-In Jurkat cells are a little tricky to handle and are very sensitive to centrifugation. You may try the following protocol to thaw the cells:

- Thaw 1 vial of cells into a T25 flask containing 5-7 mL of fresh medium (without selection). Do not spin down the cells to remove the DMSO at this point, as they are very sensitive to handling (including centrifugation). Typically, there is a lot of debris present upon thaw.
- 24 hours post-thaw, spin down the cells at 900 rpm for 2-3 minutes and resuspend gently into 5 mL of fresh medium. Spinning down the entire volume of cells greatly reduces the amount of debris in the culture.
- At day 5 or 6, it is okay to add the selection. Continue to passage the cells (spinning the cells down) every 2-3 days for a total of 1.5 weeks, each time resuspending back into only 5-7 ml of fresh medium in a T25 flask to build up the cell density. The Jurkat cells are very small and clumpy, and they expand very slowly.
- Attempt to expand the cells into a T75 flask only after about 1.5 weeks.

In theory, one can get multiple integrations of the Flp-In expression construct—an FRT-specific integration event and a random, second-site integration. However, random integration is a relatively uncommon event. Limiting the amount of DNA in the transfection will reduce the chance of second-site integration. We have transfected 293 cells (lacking the FRT site) with the pcDNA5/FRT vector and have identified one potential second-site integrant after screening over 200 clones. DNA integrations can be detected by Southern blot. A single integrant will display a single band; double: two; triple: three, etc. We have maintained a number of Flp-In expression cell lines for over four months and have not observed any loss of the Flp-In expression construct, whether hygromycin selection was maintained or not.

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