Avoiding lentiviral transduction culture induced MSC senescence.
Avoiding lentiviral transduction culture induced MSC senescence.
复制标题
避免慢病毒转导培养诱导 MSC 衰老。
DOI：
10.1111/j.1582-4934.2008.00651.x
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发表时间：
2009
影响因子：
5.3
通讯作者：
Kurre,Peter
中科院分区：
文献类型：
--
作者：
Pan,Yung-Wei;Kurre,Peter
图1（A）非贴壁鼠间充质干细胞（MSC）的慢病毒转导。将MSC以指定的感染复数（MOI）暴露于表达VSV-G假型绿色荧光蛋白（GFP）的慢病毒1小时，或者在接种16-24小时后贴壁（空心符号）或悬浮（实心符号）。(B)在无血清条件下改善了向mMSC的基因转移。将MSC从液氮中解冻，在磷酸盐缓冲盐水（PBS）中洗涤两次，重悬于培养基中，并立即以指定的MOI暴露于慢病毒载体。所有转导均在4 μ g/ml硫酸鱼精蛋白存在下，在1 ml终体积中进行1小时以上。在转导结束时，将细胞在PBS中洗涤两次，并接种在基于Iscoves的MSC扩增培养基中，如Peister [7]所述。如前所述产生逆转录病毒载体[6]。对于悬浮液中的转导，将细胞从对数期扩增培养物中胰蛋白酶化，或从液氮中的冷冻保存（5%二甲基亚砜[DMSO]）中解冻，并在PBS中洗涤。显示了多次重复测定的Δ。使用配对双尾Student t检验分析数据。小于0.05的P值被认为是显著的。
Fig. 1 (A) Lentiviral transduction of non-adherent murine mesenchymal stem cells (MSC). MSC were exposed to VSV-G pseudotyped green fluorescent protein (GFP) expressing lentivirus for 1 hr at the indicated multiplicities of infection (MOI), alternatively in adherence after plating 16–24 hrs earlier (open symbols), or in suspension (closed symbols).(B) Gene transfer to mMSC is improved under serum free conditions. MSC were thawed from liquid nitrogen, washed twice in phosphate buffered saline (PBS), resuspended in media, and immediately exposed to lentivector at the indicated MOI. All transductions were performed over 1 hr in 1ml final volume, in the presence of protamine sulphate at 4 g/ml. At the end of transduction cells were washed twice in PBS and plated in Iscoves based MSC expansion medium, as described by Peister [7]. Retrovirus vector was produced as previously described [6]. For transduction in suspension, cells were trypsinized from log-phase expansion culture, or thawed from cryopreservation (5% dimethyl sulfoxide [DMSO]) in liquid nitrogen and washed in PBS. Averages from multiple repeat determinations are shown. Data were analysed using the paired two-tailed Student’s t-test. P-values of less than 0.05 were considered significant.