• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        【专题讨论】外文文献讨论(一)---------欢迎大家积极参与、共同提高

        丁香园论坛

        4020
        本人的想法:拿出自己研究过的文献(外文),供大家提出问题,讨论、翻译。相信大家的讨论一定会碰撞出新的ideas来。(已经搜索了相关内容,应该不是重复法帖,请班主支持!)

        相互交流、享受提高!!!

        Small Interfering RNA-Mediated Gene Silencing in T
        Lymphocytes1
        Michael T. McManus, Brian B. Haines, Christopher P. Dillon, Charles E. Whitehurst,
        Luk van Parijs, Jianzhu Chen, and Phillip A. Sharp2
        Introduction of small interfering RNAs (siRNAs) into a cell can cause a specific interference of gene expression known as RNA
        interference (RNAi). However, RNAi activity in lymphocytes and in normal primary mammalian cells has not been thoroughly
        demonstrated. In this report, we show that siRNAs complementary to CD4 and CD8 specifically reduce surface expression of
        these coreceptors and their respective mRNA in a thymoma cell line model. We show that RNAi activity is only caused by a subset
        of siRNAs complementary to the mRNA target and that ineffective siRNAs can compete with effective siRNAs. Using primary
        differentiated T lymphocytes, we provide the first evidence of siRNA-mediated RNAi gene silencing in normal nontransformed
        somatic mammalian lymphocytes. The Journal of Immunology, 2002, 169: 5754–5760.
        I ntroduction of dsRNA into an organism can cause specific
        interference of gene expression (1). This phenomenon,
        known as RNA interference (RNAi),3 results from a specific
        targeting of mRNA for degradation by an incompletely characterized
        cellular machinery present in plant, invertebrate, and mammalian
        cells (2, 3). The proteins mediating RNAi are part of an
        evolutionarily conserved cellular pathway that processes endogenous
        cellular RNAs to silence developmentally important genes (4,
        5). In RNAi, the protein Dicer, an RNase III enzyme, is probably
        responsible for the processing of dsRNA into short interfering
        RNA (siRNA). Functional screens conducted in plants and worms
        have identified a number of other conserved genes participating in
        the RNAi pathway. These genes include a number of different
        helicases, a RNA-dependent RNA polymerase, an exonuclease,
        dsRNA-binding proteins, and novel genes of unknown function
        (for recent reviews, Refs. 6, 7, 8, 9, and 10).
        Mammalian RNAi was first described in mouse embryos using
        long dsRNA (11, 12). Then, following the analysis of the structure
        of the intermediate in this process, small interfering RNAs
        (siRNAs) were used to silence genes in mammalian tissue culture
        (13, 14). Most of the RNAi pathway genes discovered in plant and
        worm screens are also present in mouse and human sequence databases,
        supporting evidence that a conserved RNAi pathway exists
        in mammals. One of the more notable exceptions is the RNAdependent
        RNA polymerase gene, which has been shown to beinvolved in the amplification of the dsRNA in Caenorhabditis elegans
        (15, 16). This might imply that perpetuation of the RNAi
        response in mammals differs from that of lower organisms.
        Recent reports have demonstrated gene silencing by siRNA in
        mammalian cells (17–22). However, despite these initial reports,
        many uncertainties remain concerning the mechanism, physiologic
        relevance, and ubiquity of RNAi in mammalian cells. Although
        studies in tumor cell lines have demonstrated siRNA-mediated
        RNAi, it remains a major question as to whether primary cells
        from fresh tissues can undergo the RNAi response. Furthermore,
        little is known about the efficiency and longevity of siRNA-mediated
        RNAi gene suppression. In this report, we provide fundamental
        insight into the siRNA-mediated RNAi mechanism using a thymoma-
        derived cell line model to demonstrate for the first time the
        occurrence of RNAi in primary T lymphocytes.
        Materials and Methods
        Cell culture
        E10 is an immature double-positive thymocyte line derived from a TCR-
        and p53 double-mutant mouse of a mixed 129/Sv  C57BL/6 background
        as described (23). These cells, which proliferated vigorously, were maintained
        at a maximal concentration of 2  106 cells/ml and were propagated
        in complete medium: DMEM supplemented with 10% heat-inactivated
        FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and
        50 M 2-ME. Cell culture of primary lymphocytes: cells from the spleen
        and lymph nodes of DO11.10 TCR-transgenic mice (a generous gift from
        Dr. C. London, University of California, Davis, CA) were activated for 3
        days with 1 g/ml OVA peptide (residues 323–339) in RPMI medium
        containing 10% FBS.
        Transfection
        For electroporations, 2.5 mol dsRNA and/or 20 g of pEGFP-N3 plasmid
        (Clontech Laboratories, Palo Alto, CA) were added to prechilled 0.4-cm
        electrode gap cuvettes (Bio-Rad, Hercules, CA). E10 cells (1.5  107)
        were resuspended to 3  107 cells/ml in cold serum-free RPMI, added to
        the cuvettes, mixed, and pulsed once at 300 mV, 975 F with a Gene
        Pulser electroporator II (Bio-Rad). Cells were plated into 6-well culture
        plates containing 8 ml of complete medium and were incubated at 37°C in
        a humidified 5% CO2 chamber. Cell viability immediately after electroporation
        was typically around 60%. For cationic lipid transfections, 2 g of
        plasmid DNA and 100 nmol siRNAs were used per 106 cells, and transfection
        followed manufacturer’s recommended protocol. Transfection of
        primary lymphocytes: activated DO11.10 T cells were electroporated as
        above, except that the cells were resuspended to 6  107 cells/ml in cold
        serum-free RPMI and the pulse voltage was 310 mV. After electroporation,
        the cells were put into four wells of a 24-well plate, each containing 1 mlof RPMI supplemented with 1 ng/ml IL-2 (BioSource International, Camarillo,
        CA). siRNA oligos (Dharmacon, Lafayette, CO) used were as follows
        (sense strand is given): effective CD4 siRNA, CD4 no. 4, (sense)
        gagccauaaucucaucugadgdg, (anti-sense) ucagaugagauuauggcucdtdt; effective
        CD8 siRNA, CD8 no. 4, (sense) gcuacaacuacuacaugacdtdt, (antisense)
        gucauguaguaguuguagcdtdt; ineffective siRNAs, CD8 no. 1, (sense) gaaaa
        uggacgccgaacuudgdg, (anti-sense), aaguucggcguccauuuucdtdt; CD8 no. 2,
        (sense) cgugggacgagaagcugaadtdt, (antisense) uucagcuucucgucccacgdtdt;
        CD8 no. 3 (sense) aauuguguaaaauggcaccgcdcda, (antisense) ggcggugc
        cauuuuacacaadtdt; CD4 no. 1, (sense) ggagaccaccaugugccgadgdc, (antisense)
        ucggcacaugguggucuccdtdt; CD4 no. 2, (sense) ggcagagaaggauucu
        uucdtdt, (anti-sense) gaaagaauccuucucugccdtdt; CD4 no. 3, (sense)
        ccaccugcguccugucucadtdc, (antisense) gugguggacgcaggacagadgdt; CD4 no. 5
        (sense) ccaccugcguccugucucadtdc, (antisense) ucagaugagauuauggcucdtdt.
        Flow cytometry
        E10 cells (1  106) were washed once in FACS buffer (PBS supplemented
        with 2% FCS and 0.01% sodium azide), resuspended to 100 l, and
        stained directly with PE-conjugated anti-CD4 (clone RM4-5) or allophycocyanin-
        conjugated anti-CD8 mAbs, and in some experiments with PEor
        allophycocyanin-conjugated anti-mouse Thy-1.2 (clone 53-2.1) mAb.
        All mAbs were from BD PharMingen (San Diego, CA). The stained cells
        were washed once, then resuspended in 200 l FACS buffer containing 200
        ng/ml propidium iodide (PI). Unstained and singly stained controls were
        included in every experiment. 3A9, a T cell hybridoma line that had been
        infected with a MIGW green fluorescent protein (GFP) retrovirus was included
        when GFP expression was analyzed. Cell data were collected on a
        FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and fourcolor
        analyses (GFP, PE, PI, and allophycocyanin) were done with
        CellQuest software (BD Biosciences). All data were collected by analyses
        performed on 1  104 PI-negative events (viable cells). For the primary T
        cell studies, activated cells were analyzed as above, except that allophycocyanin-
        conjugated anti-CD4 and PE-conjugated anti-CD8 were used,
        and 5  104 PI-negative events were analyzed.
        Northern blot analysis of mRNA
        Cells were lysed in TRIzol reagent (Life Technologies, Grand Island, NY)
        and total cellular RNA was purified according to manufacturer’s instructions.
        RNA (10 g) was fractionated on a denaturing 1% formaldehyde/
        agarose gel and transferred to a nitrocellulose membrane. Blots were hybridized
        overnight with 32P-labeled CD4 (818 bp) or CD8 (596 bp) cDNA
        fragments. After washes, blots were analyzed by a PhosphorImager (Molecular
        Dynamics).
        Results
        siRNAs transiently induce silencing in murine thymocyte
        cell lines
        To study RNAi, siRNAs are typically delivered into cells by carrier-
        mediated transfection reagents. We developed an experimental
        system using a thymoma-derived cell line, E10 (23), wherein we
        studied the use of siRNAs to silence either CD4 or CD8, using
        the other marker as an internal specificity control. However, typical
        of lymphocytes, E10 is insensitive to several different cationic
        and noncationic transfection reagents and thus electroporation was
        used to introduce siRNAs. Using this method, 20% of the cell
        population expressed GFP from a transfected reporter vector.
        When CD4 or CD8 siRNAs were electroporated into E10, a
        marked reduction in surface CD4 or CD8 expression, respectively,
        occurred 36 h later. Flow cytometry analysis showed that
        most of the cells were transfected and expression levels were reduced
        5-fold below wild-type expression levels (Fig. 1A). The
        degree of reduction of CD8 was frequently more pronounced
        than that of CD4 and, in both cases, a small population of cells
        appeared to be either untransfected or not responsive to the siRNA
        treatment. In repeated experiments, typically 70–95% of the cells
        exhibited a 5-fold reduction in CD8 expression, although
        sometimes a smaller fraction of cells down-regulated CD8 to a
        greater degree (Fig. 1A).
        Elbashir et al. (13) reported that RNAi-induced silencing could
        be maintained for 2 wk in HeLa cells, although neither the extent
        of silencing nor the number of cell divisions was reported. A timecourse assay was performed in CD8 siRNA-transfected E10
        cells. GFP was included in these transfections to investigate the
        relationship between the uptake and expression of plasmid DNA
        and siRNAs. Because these experiments were transient transfections,
        cell doubling results in a decrease in GFP fluorescence intensity
        and number of GFP-positive cells (Fig. 1B, NS RNA).
        When CD8 siRNAs were cotransfected with the GFP reporter
        vector, CD8 expression, but not GFP expression, was markedly
        reduced (see Fig. 1B, 24 h). Several cell populations were evident,
        with the major CD8 silenced population displaying 5-fold reduced
        CD8 expression. The majority of cells within this population
        did not express GFP. However, cells that did express GFP
        also silenced CD8. This corresponded to 20% of the total cells,
        similar to the control GFP alone (Fig. 1B, NS siRNA, 24 h). This
        indicates that all of the cells expressing GFP also received an adequate
        level of siRNAs to silence CD8. In addition, a large fraction
        of cells incorporated biologically active levels of siRNAs andyet did not express plasmid DNA. In this experiment, time points
        were taken over a period of 6 days. At each time point, one-half of
        the cells were removed from the dish and replaced with fresh medium.
        The collected cells were stained for CD8 and analyzed by
        flow cytometry (Fig. 1). A decrease in CD8 surface expression
        was detectable at 12 h posttransfection, with maximal silencing at
        36 h. By 96 h, nearly all of the cells expressed wild-type levels of
        CD8. Thus, the RNAi effect in these T cells is a transient
        phenomena.
        In these experiments, there was a dramatic decrease in GFP
        expression over time, which was likely a result of dilution of the
        plasmid or potentially due to toxicity of high GFP expression.
        Because 100% of the GFP-expressing cells exhibited CD8 silencing,
        it was possible to monitor the “fate” of this subset of
        silenced cells. The T cells that actively underwent CD8 silencing
        continued to express GFP over the time course, to the same level
        as the control population of cells that were not transfected with
        siRNAs (compare nonspecific RNA to CD8 siRNA). At 96 h,
        5% of the total cells were GFP-positive in cells treated with
        nonspecific siRNAs and in CD8 siRNA-treated samples. These
        few remaining GFP-positive cells exhibited normal levels of
        CD8 expression. This suggests that the cells did not specifically
        undergo apoptosis as a result of siRNA transfection and subsequent
        CD8 silencing.
        Specificity of siRNA-mediated silencing
        Although the GFP transgene expression was not affected during
        CD8 silencing, the expression of endogenous genes might have
        been nonspecifically affected. To address this question, the expression
        levels of CD4 and Thy1.2 T cell markers were examined in
        cells actively undergoing CD8 silencing. Examination of these
        markers revealed that there was no reduction of nontargeted gene
        expression when compared with the control nontransfected cells
        (Fig. 2A), even over extended times (not shown). Although unlikely
        for this cell line, an additional analysis confirmed that the T
        cells did not become activated, as they do not up-regulate CD69
        (Fig. 2A). Together, these experiments confirm the specificity of
        siRNA-mediated CD8 silencing.
        Stability of targeted CD8 mRNA
        Short temporal RNAs such as lin-4 and let-7 mediate silencing by
        binding to the 3-untranslated region (UTR), thus suppressing
        translation (24–26). This is in marked contrast to the posttranscriptional
        mRNA degradation effected by siRNAs. To distinguish between
        these two potential mechanisms for CD8 silencing, a time
        course Northern blot analysis of CD8 mRNA was performed.
        The process of silencing did not appreciably affect the growth rate,
        as compared with control nonspecific siRNA transfections performed
        in parallel (not shown). Flow cytometry analysis indicated
        that the RNAi response in these cells lasted 3–4 days (8–10 cell
        doublings), which corresponds to an 100-fold increase in cell
        mass (Fig. 2B). Time course analysis was performed in four independent
        experiments and expression of CD8 was typically suppressed
        5-fold or greater.
        At various time points, a fraction of the cells was used to isolate
        total RNA for Northern blot analysis (Fig. 2C). The CD8 mRNA
        was resolved into two bands, due to alternative splicing (27, 28).
        Levels of CD8 mRNA decreased during the course of CD8
        silencing. Densitometric analysis of the CD8 mRNA bands was
        performed and normalized to the internal control CD4 band. At the
        point of maximal silencing, mRNA levels decrease only 2.5-fold.
        This value is not commensurate with the 5-fold decrease in protein
        expression determined by the flow cytometric analysis. However,
        this RNA was prepared from total cells in which 30% of the
        cells did not exhibit any silencing. When corrected for this reduction,
        CD8 mRNA was nearly proportionate to levels in reduction
        of CD8 protein. These Northern blots were performed multiple
        times with similar results. Thus, although it is clear that CD8
        mRNA decreases, we cannot rule out additional silencing phenomena
        such as cotranslational repression.
        Regional sensitivity of an mRNA to silencing by a siRNA
        A major outstanding question is whether any region of a mRNA
        can serve as an effective target for siRNA-directed silencing. Several
        different siRNAs that targeted different regions of the CD8阿尔法mRNA were tested. Of the first two CD8 siRNAs that were transfected,
        only one was active. To more quantitatively examine this
        difference, cells were transfected with varying amounts of siRNAs
        and CD8 expression was measured by flow cytometry. Cells undergoing
        silencing were quantified and compared with control
        nonspecific siRNA treatment (Fig. 3A). For the effective CD8
        siRNA, picomolar amounts were sufficient to induce some silencing
        and higher amounts produced a graded response. For the noneffective
        CD8 siRNA, even at the highest concentration tested,
        there was no activity.
        As these studies progressed, we observed that the majority of the
        synthetic CD4 and CD8 siRNAs were noneffective at silencing.
        For CD8, four different siRNAs were synthesized and tested in
        the flow cytometry assay: one overlapped the start codon, one
        which targeted the open reading frame (ORF), one which overlapped
        the stop codon, and one which targeted the 3-UTR 15 nt
        after the stop codon. Only the siRNA which targeted the 3-UTR
        15 nt after the stop codon effectively silenced CD8 expression.
        For CD4, five siRNAs were synthesized which targeted corresponding
        regions to those for the CD8 mRNA (Fig. 3B). In this
        case, only the siRNA that targeted the stop codon was effective at
        reducing CD4 expression levels. An examination of the nucleotide
        sequences did not reveal any obvious differences between the effective
        and ineffective siRNAs.
        For each of the above siRNAs, the silencing assay was performed
        at different siRNA concentrations. None of the inactive
        siRNAs generated detectable silencing at five times the highest
        concentration of the active siRNAs (Fig. 3A and data not shown).
        However, these inactive siRNAs were able to compete with the
        silencing of the active siRNAs. In these competition experiments,
        inactive CD8 siRNAs were added into the cuvettes containing the
        active CD8 siRNA, so that both could be electroporated into the
        cells simultaneously. Varying concentrations were tested, and cells
        were monitored for CD8 silencing at 36 h (Fig. 4). It was found
        that when the total siRNA pool contained an inactive CD4 or
        CD8 siRNA, then silencing mediated by an active siRNA was
        markedly reduced (Fig. 4, A and B). These results mirror the ability
        for active siRNAs to compete for other active siRNAs, a response
        that we observed for attempting silencing of both CD4 and CD8
        simultaneously (Fig. 4, C and D). The inability to silence both CD4
        and CD8 simultaneously in the same cell might suggest that
        siRNA-mediated RNAi is titratable, as has been described for silencing
        using long dsRNAs in C. elegans (29).
        To test whether the above siRNAs were also inactive in other
        cell types, the CD4 and CD8 genes were expressed from CMVdriven
        promoters in HeLa cells. The CD8 expression construct
        contained two regions that corresponded to target sites for effective
        and ineffective siRNAs in E10. In this assay, cationic lipid cotransfection
        of the mouse CD4 and CD8 plasmid vectors was
        performed with either the effective or noneffective CD8 siRNA.
        When compared with the nonspecific siRNA control, CD8-specific
        RNAi silencing was recapitulated in HeLa cells, and the ORFtargeted
        siRNA was still ineffective at silencing (Fig. 5A). These
        results suggested that the noneffective siRNA phenomenon is not
        unique to the T cell line, but is likely a feature of either the siRNA
        sequence, or more likely the mRNA. The concentration dependence
        of the effective and ineffective siRNA was evaluated in the
        HeLa cell assay. In this experiment, cationic lipid:siRNA complexes
        were preformed and added to the cells as previously described
        (13). The effective siRNA exhibited a concentration dependence;
        however, the ineffective siRNAs remained inactive even
        at the highest concentrations (Fig. 5B).
        siRNA-mediated silencing in primary mouse T cells
        To test whether primary cells are sensitive to siRNA-mediated
        silencing, the CD4/CD8 siRNAs characterized above were used
        to silence in primary mouse T cells taken from spleen. In theseognizes
        OVA peptide in the context of MHC class II were isolated
        from these mice are predominantly CD4; however, a small number
        (15%) of CD8 cells exist in these mice. Efforts to transfect
        and silence naive T cells were unsuccessful, but if the cells were
        stimulated to divide by the cognate OVA peptide, CD4 and CD8
        silencing could be accomplished similar to the E10 thymoma cell
        line. Electroporation of CD4 siRNAs into activated primary T cells
        resulted in an approximate 5-fold decrease in CD4 surface expression
        compared with an unrelated siRNA control (Fig. 6). Costaining
        for CD8 on the same cells demonstrated that the down-regulation
        of CD4 was specific. The maximal degree of silencing was
        reached at 48 h posttransfection. Later time points could not be
        collected because of reduced cell viability after 72 h in culture.
        Similarly, the subset of CD8-positive T cells electroporated with
        CD8 siRNA exhibited a maximal 3.3-fold decrease in CD8 levels.
        Furthermore, the degree of silencing in the sample population
        with the alternate coreceptor (i.e., CD4 in a CD8 siRNA-treated
        sample) verified that the RNAi response was specific (data not
        shown). These results demonstrate that primary, mature T cells are
        able to perform RNAi. The overall degree, kinetics, and specificity
        of silencing of CD4 or CD8 in primary T cells was comparable
        to that of the E10 cell line, further supporting the validity of using
        this line to characterize T cell RNAi.Discussion
        The CD4 and CD8 T cell surface glycoproteins are of central
        importance to immune function and disease. We have quantitatively
        tested the efficacy of a variety of siRNAs to suppress the
        expression of these glycoproteins. Targeting the CD4 and CD8
        markers was attractive since turnover of coreceptor message is
        fairly rapid (12 h for CD8), and changes in surface expression
        can be rapidly and easily assayed by flow cytometry. In this analysis
        of two different genes, we observed that T cells and thymocytic
        cell lines are amenable to siRNA-mediated silencing. These
        studies revealed that siRNA-mediated RNAi is transient, lasting
        approximately eight cell doublings. Not every siRNA was able to
        induce silencing, and the RNAs which targeted the 3-UTR were
        effective for both genes. Although small temporal RNAs (stRNAs)
        mediated translational repression at the mRNA 3-UTR (for recent
        reviews, see Refs. 30–34), Northern blot analysis of CD4 and
        CD8 mRNA indicated posttranscriptional degradation of the
        mRNA, consistent with a RNAi-type mechanism of silencing. Finally,
        in primary T cells, the overall penetrance and kinetics of
        CD4 and CD8 siRNA-mediated RNAi was found to be similar to
        that observed in the E10 thymoma cell line.
        In several experiments, and using electroporation, we found efficient
        uptake and silencing of 90% of the cells. However, this
        required the addition of a relatively high amount of siRNA (2.5
        mol/1.5  107 cells); Northern blot analysis indicates that only a
        fraction of the siRNAs (3  104 siRNAs/cell) become associated
        with the cells (data not shown). Only a fraction of the siRNAs that
        become associated with cells probably are functional in silencing
        gene expression. At lower concentrations of siRNAs, a similar
        fraction (70–95%) of cells exhibit a reduction in CD8 expression,
        albeit at reduced efficiency. Using either electroporation for T cells
        or Lipofectamine 2000 for HeLa cells, we found that 100% of the
        cells that take up and express a cotransfected GFP marker also
        perform RNAi. Based on this fact, it should be possible to design
        gene function experiments which enrich the pool of silenced cells
        by selecting for the activity of a transfected plasmid reporter.
        Time course analysis of CD8 silencing in the E10 cell line
        indicated that the silencing was transient in nature, lasting 3–4
        days. As this cell line doubles rapidly, this value corresponds to
        approximately eight cell doublings. Northern blots indicated that
        silencing corresponded to a reduction in mRNA levels, commensurate
        with the predicted model for RNAi. A translational repression
        mechanism has been suggested for silencing mediated by
        stRNAs via the 3 untranslated region of developmentally important
        genes. Although the reduction in mRNA level approximated
        that of CD8 expression, we cannot rule out the possibility of
        additional translational repression mechanisms.
        Only a limited number of the siRNA sequences tested could
        induce RNAi. For the silencing of most genes, on average one of
        two candidate siRNAs designed is active in contrast to the one of
        four and one in five siRNAs tested in targeting CD4 and CD8 (6).
        It is interesting to note that the siRNAs that were active in silencing
        targeted the 3-UTR and stop codon. The restrictive utilization
        of the 3-UTR siRNAs did not appear to be cell-type specific, as
        active and inactive siRNAs gave similar results in HeLa cells. It is
        unclear why targeting the mouse CD4 and CD8 mRNA 3-UTRs
        were effective for performing siRNA-mediated RNAi, while other
        sites were not. One possibility is that further testing of other
        mRNA regions would result in productive silencing (35). Alternatively,
        perhaps the 3-UTR of these genes is particularly accessible
        for targeting. Silencing of developmentally timed genes in the endogenous
        stRNA pathway is specific for the 3-UTR (25, 36). This
        could be a common feature of developmentally timed genes, because
        both CD4 and CD8 are also expressed in a developmentally
        timed manner.
        Attempting to silence both CD4 and CD8 simultaneously resulted
        in lower levels of silencing of each gene. These results
        supports a previously recognized observation that the RNAi response
        is titratable (29). Surprisingly, several of the siRNAs that
        were inactive competed for silencing when coelectroporated with
        active siRNAs. While this manuscript was in preparation, another
        group reported similar findings for the silencing of human coagulation
        trigger factor (37). However, another group has reported
        success in dual gene targeting of Lamin A/C and NuMA proteins
        in HeLa cells (38). The data presented in this study indicate that
        the inactive siRNAs are recognized by cellular processes but either
        cannot be converted to an active structure for gene silencing or
        cannot gain access to their complementary sequences on the
        target mRNA.
        This work presents the first evidence for silencing by siRNA in
        primary somatic mammalian lymphocytes. In these studies, the
        degree and kinetics of CD4 and CD8 silencing in the activated
        primary cells was similar to that of the E10 cell line. In both the
        primary cells and E10 cells the onset of maximal silencing appeared
        around three to four cell doublings, which corresponded to
        36–48 h posttransfection. In the E10 cells, 100% of the cells had
        resumed normal CD8 expression by 96 h. Because the viability of
        the primary cells began to diminish at around 60 h, it was difficult
        to determine how long the RNAi response would last past 72 h. It
        is interesting to note that the cells needed to be activated in order
        for silencing to be accomplished. This could be due to the inability
        to take up the siRNAs after electroporation, as primary T cells are
        known to be difficult to transfect with nucleic acids. It is unknown
        whether mammalian cells must be in a dividing, or “competent”,
        state to perform RNAi. Future studies of siRNA-mediated RNAi in
        primary cells are required to distinguish between these two possibilities.
        Nevertheless, these findings provide a precedent upon
        which future studies of T lymphocyte biology can be designed to
        validate function by siRNA-mediated silencing.
        这是一个关于小干扰技术在淋巴细胞的膜蛋白的上应用的文章,欢迎大家提出问题,共同讨论、共同提高!
        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序