利用基因芯片分析碱性成纤维细胞生长因子诱导脐血干细胞CD34~+与CD133~+细胞基因表达的差异

背景:迄今为止,人们利用基因芯片对碱性成纤维细胞生长因子诱导CD34~+和CD133~+干细胞分化后细胞生物学性状、功能调控、基因变化及其表达差异等方面的认识尚不足,有待深入研究.目的:利用基因芯片比较脐血CD34~+和CD133~+细胞基因表达的差异,并探讨碱性成纤维细胞生长因子体外诱导脐血CD34~+和CD133~+造血干/祖细胞分化的基因表达变化,及其对碱性成纤维细胞生长因子反应性差异.方法:利用MiniMACS免疫磁珠法从脐静脉血单个核细胞中分离出CD34~+和CD133~+干/祖细胞,经含碱性成纤维细胞生长因子、B27的DMEM/F12细胞营养液培养10～15 d,提取培养前后细胞总RNA,利用Oligo GEArray(r)芯片和GEArray表达分析软件进行芯片数据分析.流式细胞仪检测CD34~+和CD133~+造血干细胞回收率.碱性成纤维细胞生长因子诱导前后CD34~+、CD133~+细胞形态变化.变性琼脂糖凝胶电泳测定RNA浓度和纯度.基因芯片检测结果.结果与结论:①对20份脐血分别进行CD34~+和CD133~+细胞分选,分选CD34~+细胞纯度为(77.52±5.06)%,回收率为(2.74±1.59)%;CD133~+细胞纯度为(79.16±3.37)%,回收率为(1.12±0.94)%.②新分选出的CD34~+细胞呈球形,经碱性成纤维细胞生长因子培养15 d后,多数细胞形态未发现显著变化,部分细胞出现贴壁生长,可见突起和梭形细胞.CD133~+细胞呈球形,经碱性成纤维细胞生长因子培养15 d后,细胞明显扩增,由圆球形变为不规则形,部分细胞出现贴壁生长.③CD34~+干细胞培养前后总RNA提取质量分别为2 236 ng和1 796 ng;CD133~+干细胞培养前后总RNA提取质量分别为2 518 ng和2 191 ng.④在所检测的263个干细胞相关基因中,脐血CD133~+细胞与CD34~+细胞基因表达差异2倍以上的基因有10种,其中5种基因表达前者高于后者,5种基因表达前者低于后者;碱性成纤维细胞生长因子诱导培养后,CD133~+细胞有32种基因表达显著高于CD34~+细胞,在检测的263个基因中,未见低于CD34~+细胞的基因.证实脐血中新分离的CD133~+细胞和CD34~+细胞基因表达仅有少数基因存在差异;CD133~+细胞对碱性成纤维细胞生长因子的反应性显著强于CD34~+细胞,表现为细胞周期调节、信号转导和分化等基因表达增强.     

GFP在人脐血CD34~+细胞中的表达及意义

目的探讨慢病毒载体在CD34+脐血细胞(CBCs)中的基因转导效率,为基因治疗的临床应用提供关键材料。方法应用1型人类免疫缺陷病毒(HIV-1)改造而成的第三代自身失活(self-inactivating,SIN)慢病毒载体(lentiviralvector)系统,通过流式细胞术检测基因导入细胞百分比,评价该载体系统在人CBCs中的基因转导效率。结果 HIV载体的转导效率在95%以上。结论基于慢病毒载体基因转导的高效性,该载体系统可作为CD34+CBCs基因转导的极好工具。     

2型重组腺相关病毒对人脐血CD34+造血干/祖细胞的转导效率研究

为探讨 2型重组腺相关病毒 (rAAV 2 )载体能否有效地转导脐血CD34+造血干 /祖细胞 ,采用rAAV 2 /GFP感染经免疫磁珠法分离的脐血CD34+造血干 /祖细胞 ,在荧光显微镜下观察GFP的表达。结果显示 ,转导 19小时后感染复数 (MOI)为 2× 10 5时 ,CD34+细胞GFP基因的表达率为 4 3%。结论 :rAAV 2能有效地转导脐血CD34+造血干 /祖细胞。     

自身失活型慢病毒载体介导的鼠基因工程调节性T细胞的制备和特性研究

目的 制备自身失活型慢病毒载体为基础的鼠基因工程调节性T细胞(Treg细胞),检测其表型变化,并观察其增殖能力及免疫抑制能力.方法 构建含鼠Foxp3基因及内部核糖体进入位点序列(IRES)和绿色荧光蛋白基因(GFP)的双顺反子自身失活型慢病毒载体.采用脂质体转染法将慢病毒载体三质粒转染293T细胞,经共培养法感染免疫磁珠分离的小鼠CD4+CD25-T细胞,流式细胞术(FCM)检测基因转染效率及细胞Foxp3、CD25、GITR、CTLA-4的表达,CCK-8法、ELISA法检测其增殖、免疫抑制能力及细胞因子分泌的变化.结果 成功构建了慢病毒表达质粒pXZ208-IRES-GFP、pXZ208-Foxp3-IRES-GFP,包装的重组慢病毒滴度达106IU/ml.以pXZ208-IRES-GFP为阴性对照组,共培养法感染CD4+CD25-T细胞,实验组细胞Foxp3、CD25、CTLA-4、GITR表达升高.在抗CD3ε单抗、抗原呈递细胞存在条件下,实验组细胞增殖能力及IL-2、IL-4、IL-10和IFN-γ的分泌均低于阴性对照组(P<0.01);且该细胞可显著抑制CD+CD25-T细胞的增殖.结论 成功构建了自身失活型慢病毒载体介导的鼠基因工程Treg细胞;基因工程Treg细胞具有与CD4+CD25+Treg细胞相似的表型及低增殖性和免疫抑制性.     

基因工程化Treg细胞对小鼠异基因骨髓移植后急性移植物抗宿主病的作用

目的 探讨输注慢病毒载体介导的鼠基因工程调节性T淋巴细胞(Treg细胞)对小鼠异基因骨髓移植后移植物抗宿主病(GVHD)的影响.方法 利用慢病毒载体介导,将鼠Foxp3基因转导入BALB/c小鼠的CD4+CD25-T淋巴细胞,即为基因工程Treg细胞.建立小鼠异基因骨髓移植模型,在移植时联合输注基因工程Treg,通过受...     

大鼠CD4+CD25-T细胞的Foxp3基因转染及其表达

背景:调节性T细胞在维持机体免疫应答稳态和免疫耐受方面具有非常重要的作用,但外周血中CD4+CD25+调节性T细胞含量极低,且增殖能力较差.目的:以携带Foxp3基因的慢病毒EGFP+载体转染大鼠CD4+CD25- T细胞,观察其在大鼠CD4+CD25- T细胞中的表达.方法:以免疫磁珠两步法分选大鼠CD4+CD25- T细胞,用携带大鼠Foxp3基因的慢病毒载体体外转染分选的细胞,以转染Foxp3基因的CD4+CD25- T细胞为实验组,EGFP空白质粒组及CD4+CD25- T细胞为阴性对照组,CD4+CD25+ T细胞为阳性对照组.荧光显微镜和RT-PCR分别从蛋白和mRNA水平检测Foxp3基因的表达.结果与结论:成功完成了免疫磁珠的分选,获得了纯度较高的CD4+CD25-T细胞和CD4+CD25+ T细胞,细胞存活率为(94±2)%,慢病毒转染的CD4+CD25- T细胞高表达Foxp3基因.表明以携带Foxp3基因的慢病毒载体系统可有效介导Foxp3基因在大鼠CD4+CD25- T细胞中高表达.     

MHC-Ⅱ基因反义RNA导入脐血CD34^＋细胞抑制HLA-DR抗原的表达

将HLA-DRBI基因cDNA片段反向插入逆转录病毒质粒pZIP-neo SV(x)BamHI位点中,构建了HLA-DRB1基因的逆转录病毒反义RNA重组表达载体,用脂质体法导入PA317细胞。用免疫磁珠法分离、富集脐血CD34~ 细胞。将培养的病毒上清感染脐血CD34~ 细胞,筛选获得抗性克隆和进行CFU-GM的培养。用RT-PCR法从经G418选择产生的抗性克隆已证实有反义RNA目的基因的表达。流式细胞仪(FACS)检测转染的脐血细胞中HLA-DR抗原阳性率为28％(对照组为45％),其抑制率为38.2％。结果表明导入脐血细胞的MHC-Ⅱ类基因反义RNA重组体可降低其HLA-DR抗原的表达。本实验结果为用反义核酸技术在临床脐血移植中防止移植物抗宿主反应提供了新方法。     

Fn、TPO基因修饰对人骨髓间充质干细胞的影响

目的 观察纤维连接蛋白(Fn)、血小板生成素(TPO)融合基因修饰对人骨髓间充质干细胞(MSC)的影响.方法 构建携带Fn-TPO融合基因的重组逆转录病毒载体,并以其对骨髓MSC进行基因修饰;观察Fn-TPO基因在骨髓MSC中的表达,以及基因修饰后骨髓MSC的体外增殖、黏附造血细胞和分泌TPO的能力;并将脐血CD34+细胞接种到基因修饰后骨髓MSC形成的滋养层,培养7d观察修饰后骨髓MSC对造血细胞体外扩增和集落形成能力的影响.结果 成功构建携带Fn-TPO基因的重组逆转录病毒载体且以该逆转录病毒载体对骨髓MSC进行体外基因修饰;Fn、TPO基因在骨髓MSC内能够正常转录;基因修饰后的骨髓MSC体外增殖能力[(6.92±0.77)×104/ml]与对照组[(7.18±0.89)×104/ml]比较差异无统计学意义(P＞0.05);基因修饰组和对照组黏附造血细胞能力分别为0.188±0.018和0.167±0.017(P＜0.01),分泌TPO能力分别为(7.46±0.59)ng/ml和(5.58±0.37)ng/ml(P＜0.01),细胞分泌TPO的能力不受培养时间的影响,但受细胞生长状态影响;2×104脐血CD34+造血干/祖细胞经基因修饰后骨髓MSC联合必要细胞因子体外扩增7 d,有核细胞数、CD34+细胞比例、BFU-E、CFU-GM及CFU-GEMM分别为(29.9±2.7)×104、(33.3±2.8)%、109.3±4.1/1×104CD34+细胞、163.7±7.1/1×104CD34+细胞、13.3±1.5/1×104CD34+细胞,较对照组明显增加(P＜0.01).结论 Fn-TPO基因修饰能够增强骨髓MSC黏附造血细胞、分泌TPO及支持脐血CD34+细胞扩增的能力.     

胶质细胞源性神经营养因子基因修饰CD34+细胞静脉移植治疗高血压大鼠脑梗死

背景:近年来,已有实验证实经颅直接给予胶质细胞源性神经营养因子(glial cell-derived neurotrophic factor,GDNF)或携带GDNF基因病毒载体有显著减少脑梗死体积、促进神经功能恢复的疗效,但其治疗方法有创,临床应用有限.目的:观察GDNF基因转染的人脐血CD34+细胞经静脉移植对自发性高血压大鼠脑梗死的疗效,并探讨其机制.方法:分离人脐血CD34+细胞,脂质体方法转染pEGFP-GDNF质粒和pEGFP空载体至CD34'细胞制备pEGFP-GDNF-CD34+和pEGFP-CD34+细胞;制备60只雄性自发性高血压大鼠大脑中动脉栓死模型并随机分为3组:pEGFP-GDNF-CD34+细胞移植组(基因细胞组)、pEGFP-CD34+细胞移植组(单纯细胞组)、生理盐水组.改良神经功能损害评分评价神经功能恢复状况;图像分析法观察TTC染色脑梗死体积;酶联免疫法检测细胞培养液与脑组织匀浆GDNF水平,荧光显微镜及免疫组织化学检测绿色荧光蛋白标记CD34+细胞及其人神经胶质纤维酸性蛋白和人神经元核抗原表达.结果与结论:经静脉移植pEGFP-GDNF-CD34+细胞向自发性高血压大鼠脑缺血区域迁移、存活、并向神经细胞定向分化,促进神经功能恢复.GDNF基因转染CD34+细胞在脑组织存活、向神经细胞分化及对大脑神经功能保护作用优于CD34+细胞.脑组织GDNF水平可能是GDNF基因转染CD34+细胞和单纯CD34+细胞移植治疗自发性高血压大鼠局灶性脑缺血疗效差异机制之一.     

白细胞介素3基因cDNA克隆与转导及其在脐血CD34+细胞中的表达

背景:单份脐血难以满足成人造血干细胞移植的要求,需对其进行体外扩增,这不仅需要较长的时间和较高的培养条件,而且容易导致干细胞自身分化,从而影响移植效果.目的:克隆人白细胞介素3基因cDNA,构建其真核表达载体并转导脐血CD34+细胞,观察白细胞介素3的表达情况.设计、时间及地点:细胞一基因组学体外实验,于2008年在承德医学院完成.材料:健康成人外周血由承德市中心血站提供,脐血由承德市妇幼保健院提供,提供者对实验均知情同意.方法:采集健康成人外周血,应用Ficoll密度梯度法分离单个核细胞,免疫磁珠法分离脐血CD34+细胞.提取白细胞介素3 mRNA,应用RT-PCT扩增白细胞介素3 cDNA,构建真核表达载体pcDNA3/IL-3.设立2组:实验组利用基因枪技术将pcDNA3/IL-3导入脐血CD34+细胞中,对照组未行转染.主要观察指标:采用人白细胞介素3 ELISA试剂盒检测脐血CD34+细胞上清液中白细胞介素3水平.结果:扩增的白细胞介素3基因cDNA理论上应为616 bp,实际PCR产物经琼脂糖凝胶电泳后,紫外线下可见预期大小的条带,反转录合成的cDNA完整.BamH Ⅰ和XbaⅠ双酶切后,电泳可见616 bp的插入片段,与白细胞介素3基因序列相同.转染第1～7天,实验组脐血CD34+细胞上清液中白细胞介素3水平明显高于未转染对照组(t=3.46,P<0.05).结论:白细胞介素3基因cDNA克隆成功,并成功构建了真核表达质粒pcDNA3/IL-3,pcDNA3/IL-3能在脐血CD34+细胞中短期有效表达.     

Efficient transduction of human lymphocytes and CD34+ cells via human immunodeficiency virus-based gene transfer vectors

The development of gene transfer systems for the efficient transduction of human primary cells including lymphocytes and CD34+ cells is a significant step in the advancement of gene therapy and cell marking protocols. Efficient gene transfer systems also represent useful tools for basic research. Here we show that human primary lymphocytes and CD34+ cells can be efficiently transduced using a VSV-G pseudotyped HIV-1-based gene transfer system. The enhanced green fluorescent protein (EGFP) was chosen as the marker transgene, because it can be easily visualized and quantitated using fluorescence microscopy and flow cytometry, thus eliminating the need for selection or PCR to score transduction. Vectors produced with this system did not generate replication-competent retroviruses (RCRs) and efficiently transduced human cell lines (40-90%), PBMCs (60%), mobilized CD34+ cells (39%), and CD34+ cells from umbilical cord blood (60%) as measured by flow cytometry. Cells treated with AZT prior to infection did not express EGFP, ruling out passive protein or plasmid DNA transfer. This was further confirmed in methylcellulose cultures, where expression in myeloid and erythroid colonies was maintained for at least 3 weeks. In addition, this HIV-based vector was able to efficiently transduce freshly isolated, not-prestimulated CD34+ cells (70% EGFP positive) in serum-free medium. Under these same conditions, a Moloney murine leukemia virus-based vector failed to transduce not-prestimulated CD34+ cells. These characteristics make this gene transfer system an excellent choice for both basic science and possible gene therapy applications.     

Inhibition of human immunodeficiency virus replication and growth advantage of CD4+ T cells and monocytes derived from CD34+ cells transduced with an intracellular antibody directed against human immunodeficiency virus type 1 Tat.

Current clinical gene therapy protocols for the treatment of human immunodeficiency virus type 1 (HIV-1) infection involve the ex vivo transduction and expansion of CD4+ T cells derived from HIV-positive patients at a late stage in their disease (CD4+ cell count <400 cells/mm3). We examined the efficiency of transduction and transgene expression in adult bone marrow (BM)- and umbilical cord blood (UCB)-derived CD34+ cells induced to differentiate into T cells and monocytes in vitro with an MuLV-based vector encoding the neomycin resistance gene and an intracellular antibody directed against the Tat protein of HIV-1 (sFvtat1-Ckappa). The expression of the marker gene and the effects of antiviral construct on subsequent challenge with monocytotropic and T cell-tropic HIV-1 isolates were monitored in vitro in purified T cells and monocytes generated in culture from the transduced CD34+ cells. Transduction efficiencies of CD34+ cells ranged between 22 and 27%. Differentiation of CD34+ cells into T cells or monocytes was not significantly altered by the transduction process. HIV-1 replication in monocytes and CD4+ T cells derived from CD34+ cells transduced with the intracellular antibody gene was significantly reduced in comparison with the degree of HIV replication seen in monocytes and CD4+ T cells derived from CD34+ cells transduced with the neomycin resistance gene alone. Further, T cells and monocytes derived from CD34+ cells transduced with the intracellular antibody gene were demonstrated to express the sFvtat1-Ckappa transgene by RT-PCR and had a selective growth advantage in cultures that had been challenged with HIV-1. These data demonstrate that sFvtat1-Ckappa inhibits HIV-1 replication in T cells and monocytes developing from CD34+ cells and supports the continuing development of a stem cell gene therapy for the treatment of HIV-1 infection.     

Retroviral vector-mediated gene expression in human CD34+CD38- cells expanded in vitro: cis elements of FMEV are superior to those of Mo-MuLV

A novel murine stromal cell line, HESS-M28, was established, which supports the expansion of human CD34+CD38- cells more than 300-fold in vitro in the presence of human IL-3 and SCF. These cells were used in an attempt to evaluate cis-acting elements of retroviral vectors in human primitive hematopoietic cells. Cord blood cells were cultured on top of the mixed cell layers of the stromal cell line, HESS-M28, and retroviral vector-producing cells. The FMEV-type vector SF/Lyt contained the spleen focus-forming virus U3 and the MESV primer-binding site (PBS), while MO3/Lyt contained the U3 region and PBS from Mo-MuLV. After transduction by the FMEV-type and Mo-MuLV-based vectors, expression of the marker gene murine CD8 (mCD8) was examined in CD34-, CD34+, and CD34+CD38- cells. In CD34+ and CD34+CD38- cells, expression of mCD8 was higher with the FMEV-type vector, SF/Lyt, compared with the cells transduced by the Mo-MuLV-based vector MO3/Lyt, although the expression was comparable in CD34- cells. Expression of marker genes was also confirmed in long-term culture-initiating cells (LTC-ICs) and SCID-repopulating cells (SRCs).     

Development of an enhanced B-specific lentiviral vector expressing BTK: a tool for gene therapy of XLA

Further development of haematopoietic stem cell (HSC) gene therapy will depend on enhancement of gene transfer safety: ad hoc improvement of vector design relating to each particular disease is thus a crucial issue for HSC gene therapy. We modified a previously described lentiviral vector by adding the Emumar B-specific enhancer to a human CD19 promoter-derived sequence (Mol Ther 2004;10:45-56). We thus significantly improved the level of expression of the green fluorescent protein (GFP) reporter gene while retaining the specificity of expression in B-cell progeny of transduced human CD34+ progenitor cells obtained from cord blood or adult bone marrow. Indeed, GFP was strongly expressed from early medullary pro-B cells to splenic mature B cells whereas transgene expression remained low in transduced immature progenitors as in myeloid and T-lymphoid progeny retrieved from xenografted NOD/SCID/gammac(null) mice. Using this lentiviral vector, we further demonstrated the possibility to express a functional human BTK protein in long-term human CD34+ cell B-lymphoid progeny. This newly designed lentiviral vector fulfils one of the pre-requisites for the development of efficient and safe gene therapy for X-linked agammaglobulinaemia, the most common primary humoral immunodeficiency disorder.     

Gene transfer to repopulating human CD34+ cells using amphotropic-, GALV-, or RD114-pseudotyped HIV-1-based vectors from stable producer cells.

A novel, stable human immunodeficiency virus type 1 vector packaging system, STAR, was tested for its ability to transduce human cord blood CD34+ progenitor cells assayed both in vitro and after transplantation into NOD/SCID mice. Vectors pseudotyped with three different gammaretrovirus envelopes were used: the amphotropic MLV envelope (MLV-A), a modified gibbon ape leukemia virus envelope (GALV+), and a modified feline endogenous virus RD114 envelope (RDpro). Gene transfer to freshly thawed CD34+ cells in the absence of cytokines was very low. Addition of cytokines increased gene transfer efficiency significantly and this was further augmented if the cells were prestimulated for 24 h. Concentration of the vectors (15-fold) by low-speed centrifugation increased gene transfer to CD34+ cells in vitro even further. More than 90% of cells were transduced with a single exposure to the RDpro vector as determined by GFP expression using flow cytometry. The two other pseudotypes transduced approximately 65-70% of the cells under the same conditions. Transplantation of CD34+ cells prestimulated for 24 h and then transduced with a single exposure to concentrated vector revealed that the RDpro vector transduced 55.1% of NOD/SCID repopulating human cells, which was significantly higher than the MLV-A (12.6%)- or GALV+ (25.1%)-pseudotyped vectors.     

Efficient gene transfer into human cord blood CD34+ cells and the CD34+CD38- subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV

Recombinant adeno-associated viral (rAAV) vectors have been evaluated for their ability to transduce primitive hematopoietic cells. Early studies documented rAAV-mediated gene expression during progenitor derived colony formation in vitro, but studies examining genome integration and long-term gene expression in hematopoietic cells have yielded conflicting results. Such studies were performed with crude vector preparations. Using improved methodology, we have generated high titer, biologically active preparations of rAAV free of wild-type AAV (less than 1/107particles) and adenovirus. Transduction of CD34+ cells from umbilical cord blood was evaluated with a bicistronic rAAV vector encoding the green fluorescent protein (GFP) and a trimetrexate resistant variant of dihydrofolate reductase (DHFR). Freshly isolated, quiescent CD34+ cells were resistant to transduction (less than 4%), but transduction increased to 23 +/- 2% after 2 days of cytokine stimulation and was further augmented by addition of tumor necrosis factor alpha (51 +/- 4%) at a multiplicity of infection of 106. rAAV-mediated gene expression was transient in that progenitor derived colony formation was inhibited by trimetrexate. Primitive CD34+ and CD34+, CD38- subsets were sequentially transduced with a rAAV vector encoding the murine ecotropic receptor followed by transduction with an ecotropic retroviral vector encoding GFP and DHFR. Under optimal conditions 41 +/- 7% of CD34+ progenitors and 21 +/- 6% of CD34+, CD38- progenitors became trimetrexate resistant. These results document that highly purified rAAV transduce primitive human hematopoietic cells efficiently but gene expression appears to be transient. Gene Therapy (2000) 7, 183-195.     

Human cord blood CD34+CD38- cell transduction via lentivirus-based gene transfer vectors.

The efficient transfer and sustained expression of a transgene in human hematopoietic cells with in vivo repopulating potential would provide a significant advancement in the development of protocols for the treatment of hematopoietic diseases. Recent advances in the ability to purify and culture hematopoietic cells with the CD34+CD38- phenotype and with in vivo repopulating potential from human umbilical cord blood provide a direct means of testing the ability of transfer vectors to transduce these cells. Here we demonstrate the efficient transduction and expression of enhanced green fluorescent protein (EGFP) in human umbilical cord-derived CD34+CD38- cells, without prestimulation, using a lentivirus-based gene transfer system. Transduced CD34+CD38- cells cultured in serum-free medium supplemented with SCF, Flt-3, IL-3, and IL-6 maintained their surface phenotype for 5 days and expressed readily detectable levels of the transgene. The average transduction efficiency of the CD34+CD38- cells was 59 +/- 7% as determined by flow cytometry. Erythroid and myeloid colonies derived from transduced CD34+CD38- cells were EGFP positive at a high frequency (66 +/- 9%). In contrast, a murine leukemia virus-based vector transduced the CD34+CD38- cells at a low frequency (<4%). These results demonstrate the utility of lentiviral-based gene transfer vectors in the transduction of primitive human hematopoietic CD34+CD38- cells.     

Distinct effect of retroviral-mediated IFN-alpha gene transfer on human erythroleukemic and CD34+ cell growth and differentiation

Human interferon-alpha (IFN-alpha) has been used in the management of leukemia, but its diverse adverse effects may influence the ability of IFN-alpha to treat this disease. We constructed two retroviral vectors, LSN-IFN-alpha and LNC-IFN-alpha, in which IFN-alpha cDNA was driven by viral LTR and CMV promoters, respectively. After transduction into the PA317 and PG13 retroviral packaging cells, high titers of retrovirus were produced and were used to infect K562 and human BM CD34+ hematopoietic cells. The IFN-alpha gene expression in transduced K562 cells was confirmed by Northern blot, RT-PCR, RIA, and biologic assay. Cell proliferation and cell viability in IFN-alpha-transduced K562 cells were significantly suppressed as compared with control K562 cells. Although the IFN-alpha expression in K562 cells did not affect BCR/ABL expression, it apparently upregulated the production of adhesion molecules (VLA-4 and Mac-1). We evaluated the effect of IFN-alpha gene transfer on human CD34+ cells infected with LSN-IFN-alpha retrovirus with the aid of fibronectin (FN) fragment CH-296 and growth factors. RIA showed that IFN-alpha-transduced CD34+ cells produced 72.2+/-15 U/ml of IFN-alpha compared with 4.3+/-1.2 U/ml in control CD34+ cells. Methylcellulose clonogenic assay indicated that IFN-alpha-transduced CD34+ cells produced similar numbers of burst-forming units-erythrocytes (BFU-E)/colony-forming units-GM (CFU-GM) colonies as compared with control CD34+ cells. Selected colonies expressed IFN-alpha and neo(r) mRNA, as measured by RT-PCR. These studies indicate that retrovirus-mediated IFN-alpha gene transfer may provide a useful tool for studying the effect of IFN-alpha gene transfer on leukemic cells and long-lived CD34+ cells.