共查询到20条相似文献，搜索用时 31 毫秒
1.
OsEBP-89基因是水稻中一个EREBP(ethyleneresponsive elements binding protein)类转录因子.Northern杂交检测出OsEBP-89基因的表达受激素及其类似物ACC、2,4-D、ABA、BR、JA、GA和6-BA的诱导,此外也受盐胁迫的诱导.通过序列比较在OsEBP-89基因启动子区找到一个类似乙烯应答元件(ethvlene responsive element,ERE),称为IVC box.实验表明IVC box能够和水稻未成熟胚乳核蛋白特异性结合;用IVC box和35S(-46)mini-promoter/GUS的融合结构转化水稻,在转基因水稻愈伤细胞中GUS基因的表达能被ACC诱导.然而,OsEBP-89基因5′调控区失去了IVC box及其上游序列后,受ACC诱导而表达的能力有所降低,但降低的幅度不大.推测OsEBP-89基因可能有不止一个受乙烯调控的位点. 相似文献
2.
We used translation-blocking morpholinos to reduce protein levels in Giardia intestinalis. Twenty-four hours after electroporation with morpholinos targeting either green fluorescent protein or kinesin-2b, levels of these proteins were reduced by 60%. An epitope-tagged transgene can also be used as a reporter for morpholino efficacy with targets lacking specific antibodies.Giardia intestinalis (synonym Lamblia) is a parasitic protist and a major cause of diarrheal disease in developing countries (1, 16). Certain aspects of giardial biology have proved intractable for researchers seeking to study gene function. The trophozoite contains two diploid nuclei, making the cell effectively tetraploid and gene knockouts infeasible (1a). In addition, although Giardia contains RNA interference genes (homologs of Dicer and Argonaute genes) and this machinery was recently implicated in the control of antigenic variation (13, 15), attempts to manipulate this system for gene knockdown have been unsuccessful (C. C. Wang, personal communication).While many valuable tools for studying gene function in Giardia have been developed, a fast, reliable method to knock down genes is still lacking. A few researchers have used virus-mediated ribozyme constructs to achieve gene knockdown (3). However, the selection of transformants may eliminate cells in which knockdown is deleterious. Dominant-negative mutants are also used to study gene function (4, 7), but few genes are amenable to this approach. Various levels of knockdown (from 34 to 100%) have been achieved by expressing the antisense sequence of large portions of the open reading frame of the target gene under the control of a strong promoter (6, 9, 10, 13, 20). But because a promoter allowing for the tight control of Giardia gene expression has not been developed, this approach can be applied to study only nonessential (13) or encystation-specific (6, 10) genes. Furthermore, it is not possible to control for off-target effects when using this technique.Morpholinos are modified antisense oligonucleotides in which a six-membered morpholine ring replaces the deoxyribose ring of DNA and nonionic phosphorodiamidate linkages replace the typical anionic phosphodiester linkages (11). As a result, they cannot be degraded by cellular nucleases and are stable in cell culture (19). When designed to bind between the 5′ cap and a point 25 nucleotides downstream of the translation start site of the target mRNA, morpholinos (typically 25-mers) will sterically block ribosome binding and prevent the translation of the target gene (19). These translation-blocking morpholinos have been used previously to prevent new protein synthesis in trypanosomes (17).To determine the efficacy of translation-blocking morpholinos in Giardia, we first targeted enhanced green fluorescent protein (enhanced GFP) in a strain expressing the GFP gene under the control of the glutamate dehydrogenase promoter (23). In this strain, diffuse GFP fluorescence is found throughout the cytoplasm (data not shown). The 25-mer morpholino was designed to target the first 24 bases of the GFP open reading frame, plus 1 base upstream of the start codon (Table (Table1).1). As a specificity control, we used a morpholino containing five mispaired bases (Table (Table1).1). The inclusion of five mispairs has been shown to destabilize the pairing of the morpholino with its target unless the cytoplasmic morpholino concentration is extremely high; therefore, this control can act as a sensor for concentration-dependent off-target effects (11). This control morpholino also shares its chemical properties and base composition with the experimental morpholino.
Open in a separate windowaLowercase letters indicate mispaired bases.Giardia trophozoites were cultured as described previously (14), and the introduction of morpholinos by electroporation was done essentially as described previously for plasmids (18). Lyophilized morpholinos (Gene Tools, LLC, Philomath, OR) were resuspended in sterile water to a concentration of 1 mM. This stock was added directly to a 0.4-cm cuvette with ∼5 × 106 cells in 0.3 ml of medium to produce the desired concentration of morpholinos. For the negative control, a volume of sterile water equal to the volume of the morpholino suspension was added. After electroporation, cells were grown for the amounts of time indicated in the figures and then analyzed by flow cytometry (Fig. (Fig.1).1). For flow cytometry, cells were first incubated in warm HEPES-buffered saline for 30 min to facilitate GFP fluorescence and then fixed with 1% paraformaldehyde and counted on a Beckman-Coulter EPICS XL analyzer. Twenty thousand cells from each sample were counted, enhanced GFP fluorescence was measured, and a gate for GFP-positive cells was created based on comparison to wild-type cells (see Fig. S1 in the supplemental material).Open in a separate windowFIG. 1.Time course of GFP knockdown by morpholinos. Cells were collected at the indicated times after electroporation with water (no-morpholino control), 100 μM mispair anti-GFP morpholino, 10 μM anti-GFP morpholino, or 100 μM anti-GFP morpholino. Fixed samples were subjected to flow cytometry and categorized as GFP positive or GFP negative compared to a wild-type control (see Fig. S1 in the supplemental material). The number of GFP-positive cells in the no-morpholino control culture at each time point was set to 100%. The 96-h culture consisted of cells that were passaged 72 h after electroporation and then grown an additional 24 h before collection. These data are the averages of results for three biological replicates, and error bars represent one standard deviation.In all cases, the presence of a morpholino had no observable effect on cell growth compared to that of the no-morpholino control (data not shown). GFP levels in the no-morpholino and mispair controls remained approximately equal at all time points (Fig. (Fig.1;1; also see Fig. S2 in the supplemental material for representative flow cytometry histograms). However, 24 h after electroporation, GFP levels in the cultures treated with 100 μM anti-GFP morpholino had decreased by ∼60%, and they remained at approximately this level for the next 2 days, increasing only after the cultures were passaged and allowed to grow for 24 h (Fig. (Fig.1,1, 96 h). In the cultures treated with 10 μM anti-GFP morpholino, protein levels decreased by a maximum of ∼40%. Treatment with a higher concentration of morpholino (200 μM) produced approximately the same level of knockdown as treatment with 100 μM (see Fig. S3 in the supplemental material). For both the GFP and G. intestinalis kinesin-2b (GiKIN2b) experiments described below, maximum knockdown was not achieved until 24 h after electroporation (data not shown). This delay likely reflects the time needed for the turnover of preexisting protein in the cell and/or dilution by cell division.To determine whether the remaining GFP-positive cells received morpholinos, we treated cells with a fluorescently labeled anti-GFP morpholino by electroporation. Twenty-four hours after electroporation, >99% of cells with 100 or 200 μM morpholino were positive for morpholino fluorescence whereas only ∼37% of cells with 10 μM morpholino were positive (see Fig. S4 in the supplemental material). However, within the morpholino-positive cells, there was no obvious correlation between morpholino fluorescence and GFP levels. The remaining GFP-positive cells in these experiments likely started out with more GFP than the GFP-negative cells (the population is heterogeneous due to variations in plasmid copy number) and did not receive enough morpholinos to reduce the GFP below the level of detection.Next, we designed a morpholino to target an endogenous protein, G. intestinalis KIN2b (GiKIN2b) (Table (Table1).1). This protein is a homolog of the previously characterized G. intestinalis kinesin-2a (GiKIN2a), which is involved in anterograde intraflagellar transport in Giardia (7). In the GiKIN2a study, the expression of a dominant-negative GiKIN2a mutant produced cells with shortened flagella (7). Because the two kinesin-2 homologs likely function in the same complex (21), we chose to target GiKIN2b for morpholino knockdown to compare the resulting phenotype with the known phenotype of the GiKIN2a dominant-negative mutant.To track the levels of the GiKIN2b protein, we produced a GiKIN2b-specific antibody that recognizes a single ∼72-kDa band in Giardia extracts (Fig. (Fig.2A).2A). A 732-nucleotide segment of GiKIN2b (GenBank accession no. XP_001708236) containing the region encoding the C-terminal stalk and tail of the protein was cloned from Giardia genomic DNA by using primers kin-2b forward (5′-ACTGATATCTAATGGGTGCAGGGTTTACGGGCTATAC-3′) and kin-2b reverse (5′-ACTGCGGCCGCTCAACCGAAACCAGCCATGCCACG- 3′) and introduced into the vector pET30c (EMD Biosciences, Gibbstown, NJ), which includes a sequence encoding an N-terminal His tag. Purification on Ni-nitrilotriacetic acid beads under denaturing conditions was performed according to the instructions of the bead manufacturer (Qiagen, Valencia, CA). The purified protein was used to inoculate two New Zealand White rabbits according to a 77-day immunization protocol developed by Covance (Denver, PA).Open in a separate windowFIG. 2.Translation-blocking morpholinos reduce GiKIN2b protein levels and cause extreme shortening of the flagella. (A) The GiKIN2b antibody produced for this study recognizes a band of the correct size (72 kDa) on Giardia extract immunoblots. (B) Results from a representative immunoblot analysis of samples 24 h after electroporation with water (no-morpholino control), 100 μM mispair anti-GiKIN2b morpholino, or 100 μM anti-GiKIN2b morpholino. PDI-2 was used as a loading control. For each sample, the ratio of GiKIN2b to PDI-2 is given. (C) Average ratios of GiKIN2b to PDI-2 on immunoblots from three experiments like the one described in the legend to panel B. The GiKIN2b/PDI-2 ratio in the no-morpholino control was set to 100%. Error bars represent one standard deviation from the average for three biological replicates. (D) Diagram of a Giardia cell and immunolabeling of wild-type and anti-GiKIN2b morpholino-treated cells. Fixed cells were labeled with an anti-α-tubulin antibody. In the diagram, the identities of the four pairs of flagella are indicated: afl, anterior flagella; pfl, posteriolateral flagella; vfl, ventral flagella; and cfl, caudal flagella. The median body (mb), a bundle of microtubules with an unknown function; the ventral disc (vd), used to attach to substrates; and two nuclei (N) are also labeled. Scale bar, 11 μm. (E) Distribution of mutant phenotypes in fixed and immunolabeled samples. Cells were collected 24 h after electroporation with water (no-morpholino control), 100 μM mispair anti-GiKIN2b morpholino, or 100 μM anti-GiKIN2b morpholino. Cells were classified as normal, mutant (missing the external portions of at least two pairs of flagella), or other (could not be categorized). One hundred cells from each sample were counted.The resulting antibody was used on fluorescent immunoblots at a concentration of 1:50,000, along with an anti-protein disulfide isomerase 2 (anti-PDI-2) antibody (a gift from F. D. Gillin [8]) at 1:500,000 as a loading control (9) (Fig. (Fig.2B).2B). The ratio of GiKIN2b to PDI-2 was used to compare amounts of GiKIN2b at different time points (Fig. 2B and C). The blots were visualized with a Li-Cor Odyssey infrared imager, and densitometry was performed using the Li-Cor Odyssey software according to the manufacturer''s instructions.When used at 100 μM, the anti-GiKIN2b morpholino achieved a 60% reduction in protein levels in 24 h (Fig. (Fig.2C).2C). Mutants with disrupted cytoskeletons were also observed among fixed cells (Fig. (Fig.2D).2D). Immunostaining with the TAT1 antibody (22) (a gift from K. Gull), image collection, and deconvolution were performed essentially as described previously (12). The cytoskeleton of a Giardia cell includes four pairs of flagella, a bundle of microtubules called the median body, and several other structures (5) (Fig. (Fig.2D).2D). In the mutant cells, the entire external regions of at least two pairs of flagella were missing, with only internal axonemes remaining. The caudal and posteriolateral flagella were most often affected, though three or even all four sets of flagella of some cells were affected. These cells also often had reduced or missing median bodies. After 24 h, approximately 14% of cells in the knockdown cultures displayed mutant phenotypes, compared to 5% in the mispair control culture and 1% in the no-morpholino control culture (Fig. (Fig.2E2E).When using translation-blocking morpholinos in Giardia, determining the level of knockdown generally requires a specific antibody. However, zebrafish researchers have used an epitope-tagged protein to provide a readout for morpholino efficacy (2). To determine whether this strategy would work for Giardia, we used the GiKIN2b morpholino in a strain carrying a plasmid encoding a C-terminally GFP-tagged GiKIN2b protein under the control of its native promoter (7). Thus, the same morpholino could be used to knock down the endogenous and GFP-tagged GiKIN2b proteins simultaneously.Although the endogenous protein in this strain was reduced to the same level as that in the wild type after 24 h, GiKIN2b-GFP took a total of 48 h after electroporation to reach that level (Fig. (Fig.3A).3A). This delay may be due to a difference in the turnover rate between the endogenous GiKIN2b and the GFP-tagged protein and probably also to high gene dosages in cells with multiple copies of the plasmid. Twenty-four hours after electroporation, 50% of the morpholino-treated cells displayed mutant phenotypes (shortened/missing flagella) (Fig. (Fig.3B).3B). In fact, even 15% of the cells of the GiKIN2b::GFP strain without morpholino treatment were mutants. We hypothesize that the GiKIN2b-GFP fusion protein interferes with wild-type GiKIN2b complex function, sensitizing the cells to morpholino knockdown of the remaining GiKIN2b.Open in a separate windowFIG. 3.GFP-tagged GiKIN2b is knocked down by the anti-GiKIN2b morpholino. (A) The ratios of GiKIN2b or GiKIN2b-GFP to PDI-2 on immunoblots are plotted, with the ratio in the mispair control sample set to 100%. Cells were collected 24 and 48 h after electroporation with 100 μM mispair anti-GiKIN2b morpholino or 100 μM anti-GiKIN2b morpholino. (B) Distribution of mutant phenotypes in fixed and immunolabeled samples. GiKIN2b::GFP cells were collected 24 h after electroporation with water (control) or 100 μM anti-GiKIN2b morpholino. Cells were classified as normal, mutant (missing the external portions of at least two pairs of flagella), or other (could not be categorized). One hundred cells from each sample were counted.The phenotype of GiKIN2b knockdown reveals some aspects of kinesin-2 function that the dominant-negative GiKIN2a may have obscured. Induction of the dominant-negative GiKIN2a resulted in a 15 to 30% decrease in flagellar length (7). However, morpholino knockdown of GiKIN2b produced cells missing the entire external regions of their flagella, suggesting that the remaining external flagellar regions of the dominant-negative cells were due probably to residual activity of the wild-type heterotrimeric complex. In any case, some conclusions can be drawn from the results of both studies: that different pairs of flagella are not equally susceptible to kinesin-2 disruption and that the cytoplasmic regions of the flagella are not maintained solely by kinesin-2-mediated intraflagellar transport.Using translation-blocking morpholinos, we achieved a 60% reduction in protein levels in 24 h for both targets. However, for two different targets, unpublished data suggest that up to 80% knockdown can be achieved (A. R. Paredez and S. C. Dawson, unpublished data). Due to their efficacy, rapid action, specificity, and stability in cell culture, we believe that morpholinos have the potential to become a powerful new tool in the field of Giardia biology. 相似文献
TABLE 1.
Morpholinos used in this study| Morpholino | Sequence (5′ to 3′)a |
|---|---|
| Anti-GFP | CAGCTCCTCGCCCTTGCTCACCATG |
| Mispair anti-GFP | CAcCTgCTCGCCgTTGCTgACgATG |
| Anti-GiKIN2b | GCCTTTGCCCTTACTCTTGCTCATC |
| Mispair anti-GiKIN2b | GCgTTTcCCCTTAgTCTTcCTgATC |
3.
仅基于RNA元件构建可诱导哺乳动物细胞基因表达的调控系统 总被引：1，自引：0，他引：1
刁勇 《中国生物工程杂志》2011,31(12):120-125
大量的临床前和临床研究结果已表明基因治疗是理想的疾病治疗手段,然而如何实现治疗基因表达的精确调控仍然是研究人员面临的主要挑战。目前临床前研究常用的基因调控系统多基于控制转录,对反式转录激活因子和专门启动子元件的依赖限制了该系统的临床应用。最近,仅采用RNA元件构建的几种基因表达调控系统得到开发,其作用机制为核酶介导的RNA自我切割、RNA干扰、mRNA翻译启动或终止控制等。该类系统的调控活性由小分子配体反式控制,诱导基因表达的变化幅度可观,反应快速,在哺乳动物体内外均可实现。该系统结构模块化,调控活性可调节,可以克服现有转录调节系统的一些应用局限,对将来基因治疗的临床应用具有重要意义。 相似文献
4.
5.
诱导型一氧化氮合酶(iNOS)基因表达的调控 总被引：11，自引：0，他引：11
一氧化氮（ＮＯ）自由基有多方面的生物学功能。随着研究的深入,发现ＮＯ能与超氧阴离子（Ｏ－２·）反应生成激发态亚硝酸（ＯＮＯＯＨ＊）,它与靶分子能产生羟自由基（·ＯＨ）和二氧化氮（ＮＯ２）样反应,在体内原先认为的一些ＮＯ效应,现在知道主要是由于ＯＮＯＯ... 相似文献
6.
为构建修复突变绿色荧七蛋白（GFP）基因的反式剪接核酶,分别构建包含突变的GFP基因的XYQ5／10-pGEM重组质粒、XYQ5／10—pEGFP—C2重组质粒及用于修复该突变基因的反式剪接核酶载体trans—rib—CMV2。通过对体外共转录XYQ5／10—pGEM和trans—rib—CMV2重组质粒的RNA产物进行RT—PCR检测核酶细胞外剪接效果;通过XYQ5／10-pEGFP-C2和trans—rib—CMV2重组质粒共转染HeLa细胞检测核酶细胞内的剪接效果。结果显示,XYQ5／10—pGEM、XYQ5／10-pEGFP-C2及trans—rib—CMV2重组质粒构建成功,反式剪接核酶在细胞外及细胞内都可以修复突变基因。虽然效率不高,但为今后更大规模地研究设计反式剪接核酶打下了基础。 相似文献
7.
目的：分析FHL2基因在健康人群个体间表达的差异,评估FHL2作为一种新的辐射生物剂量计的可行性。方法：收集20例健康人血液样本,提取白细胞RNA进行反转录,以β-actin作为内参,利用实时定量PCR方法,检测人群中FHL2基因相对表达水平,并分析其差异。结果：FHL2和β-actin基因的实时定量PCR熔解曲线均为单峰,所得到的Ct值与相应的PCR产物呈良好的线性关系;20例血液标本中FHL2表达水平之间存在较大差异,且其表达水平与性别无关。结论：公认的放射诱导基因FHL2可能不适合作为辐射生物剂量计。 相似文献
8.
9.
10.
Developing tTA Transgenic Rats for Inducible and Reversible Gene Expression 总被引：1，自引：0，他引：1 
下载免费PDF全文
下载免费PDF全文 Hongxia Zhou Cao Huang Min Yang Carlisle P Landel Pedro Yuxing Xia Yong-Jian Liu Xu Gang Xia 《International journal of biological sciences》2009,5(2):171-181
To develop transgenic lines for conditional expression of desired genes in rats, we generated several lines of the transgenic rats carrying the tetracycline-controlled transactivator (tTA) gene. Using a vigorous, ubiquitous promoter to drive the tTA transgene, we obtained widespread expression of tTA in various tissues. Expression of tTA was sufficient to strongly activate its reporter gene, but was below the toxicity threshold. We examined the dynamics of Doxycycline (Dox)-regulated gene expression in transgenic rats. In the two transmittable lines, tTA-mediated activation of the reporter gene was fully subject to regulation by Dox. Dox dose-dependently suppressed tTA-activated gene expression. The washout time for the effects of Dox was dose-dependent. We tested a complex regime of Dox administration to determine the optimal effectiveness and washout duration. Dox was administered at a high dose (500 μg/ml in drinking water) for two days to reach the effective concentration, and then was given at a low dose (20 μg/ml) to maintain effectiveness. This regimen of Dox administration can achieve a quick switch between ON and OFF statuses of tTA-activated gene expression. In addition, administration of Dox to pregnant rats fully suppressed postnatal tTA-activated gene expression in their offspring. Sufficient levels of Dox are present in mother's milk to produce maximal efficacy in nursing neonates. Administration of Dox to pregnant or nursing rats can provide a continual suppression of tTA-dependent gene expression during embryonic and postnatal development. The tTA transgenic rat allows for inducible and reversible gene expression in the rat; this important tool will be valuable in the development of genetic rat models of human diseases. 相似文献
11.
Inducible Gene Expression by Nonculturable Bacteria in Milk after Pasteurization 总被引：3，自引：0，他引：3 下载免费PDF全文
下载免费PDF全文 Thusitha S. Gunasekera Anders Srensen Paul V. Attfield Sren J. Srensen Duncan A. Veal 《Applied microbiology》2002,68(4):1988-1993
The viability of bacteria in milk after heat treatments was assessed by using three different viability indicators: (i) CFU on plate count agar, (ii) de novo expression of a gfp reporter gene, and (iii) membrane integrity based on propidium iodide exclusion. In commercially available pasteurized milk, direct viable counts, based on dye exclusion, were significantly (P < 0.05) higher than viable cell counts determined from CFU, suggesting that a significant subpopulation of cells in pasteurized milk are viable but nonculturable. Heating milk at 63.5°C for 30 min resulted in a >4-log-unit reduction in the number of CFU of Escherichia coli and Pseudomonas putida that were marked with lac-inducible gfp. However, the reduction in the number of gfp-expressing cells of both organisms under the same conditions was <2.5 log units. These results demonstrate that a substantial portion of cells rendered incapable of forming colonies by heat treatment are metabolically active and are able to transcribe and translate genes de novo. 相似文献
12.
Brian Sauer 《Methods (San Diego, Calif.)》1998,14(4):381-392
Molecular techniques now allow the design of precise genetic modifications in the mouse. Not only can defined nucleotide changes be engineered into the genome of the mouse, but genetic switches can be designed to target expression or ablation of any gene (for which basic molecular information is available) to any tissue at any defined time. These strategies promise to contribute substantially to an increased understanding of individual gene function in development and pathogenesis. A powerful tool, both for the design of such genetic switches and for speeding the creation of gene-modified animals, is the Cre site-specific DNA recombinase of bacteriophage P1. Precise DNA rearrangements and genetic switches can be efficiently generated in a straightforward manner using Cre recombinase. In conjunction with inducible systems for controlling Cre expression and function, these recombination-based strategies are likely to have a profound impact on developmental biology and the generation of useful animal models of human disease. 相似文献
13.
Dharmendra K. Singh Tatiana N. Laremore Philip B. Smith Siela N. Maximova Timothy W. McNellis 《PloS one》2012,7(10)
Fibrillin4 (FBN4) is a protein component of plastoglobules, which are antioxidant-rich sub-compartments attached to the chloroplast thylakoid membranes. FBN4 is required for normal plant biotic and abiotic stress resistance, including bacterial pathogens, herbicide, high light intensity, and ozone; FBN4 is also required for the accumulation of osmiophilic material inside plastoglobules. In this study, the contribution of FBN4 to plastoglobule lipid composition was examined using cultivated apple trees in which FBN4 gene expression was knocked down using RNA interference. Chloroplasts and plastoglobules were isolated from leaves of wild-type and fbn4 knock-down trees. Total lipids were extracted from chloroplasts and plastoglobules separately, and analyzed using liquid chromatography-mass spectrometry (LC–MS). Three lipids were consistently present at lower levels in the plastoglobules from fbn4 knock-down apple leaves compared to the wild-type as determined by LC-MS multiple ion monitoring. One of these species had a molecular mass and fragmentation pattern that identified it as plastoquinone, a known major component of plastoglobules. The plastoquinone level in fbn4 knock-down plastoglobules was less than 10% of that in wild-type plastoglobules. In contrast, plastoquinone was present at similar levels in the lipid extracts of whole chloroplasts from leaves of wild-type and fbn4 knock-down trees. These results suggest that the partitioning of plastoquinone between the plastoglobules and the rest of the chloroplast is disrupted in fbn4 knock-down leaves. These results indicate that FBN4 is required for high-level accumulation of plastoquinone and some other lipids in the plastoglobule. The dramatic decrease in plastoquinone content in fbn4 knock-down plastoglobules is consistent with the decreased plastoglobule osmiophilicity previously described for fbn4 knock-down plastoglobules. Failure to accumulate the antioxidant plastoquinone in the fbn4 knock-down plastoglobules might contribute to the increased stress sensitivity of fbn4 knock-down trees. 相似文献
14.
Retro Tet基因表达系统是一种新型的高效、稳定、无毒、具有严密开 关功能的可诱导性真核细胞基因表达系统 .该系统兼备了逆转录病毒基因表达系统和Tet off Tet on基因表达系统的优点 ,在稳定表达细胞系的筛选、基因的表达与调控及基因功能研究等方面得到了成功的应用 ,同时也为基因治疗提供了一种理想的基因载体系统 相似文献
15.
采用重叠延伸PCR方法合成阻遏蛋白的编码基因tetr和插入操纵基因teto的Ketogulonigenium vulgare山梨糖脱氢酶启动子psndhteto的基因序列,借助宽宿主质粒p BBR1MCS-5,构建四环素诱导表达的穿梭质粒,转化Ketogulonigenium vulgare,获得阳性重组菌株,实现卡那霉素抗性的调控表达,结果表明:培养重组菌株2小时后,添加0.4μg/ml的四环素诱导剂后,能够在含有卡那霉素的培养基中生长,不添加四环素诱导剂的重组菌株不能在含卡那霉素的培养基中生长,确定了最适四环素的诱导浓度为0.6μg/ml。 相似文献
16.
Anna Brestovitsky Rakefet Sharf Tamar Kleinberger 《Journal of visualized experiments : JoVE》2012,(68)
Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal. 相似文献
17.
Teresa Cruz‐Bustos Srinivasan Ramakrishnan Ciro D. Cordeiro Michael A. Ahmed Roberto Docampo 《The Journal of eukaryotic microbiology》2018,65(3):412-421
Generation of conditional mutants in Trypanosoma brucei can be done by the use of RNA interference (RNAi). However, RNAi frequently produces off target effects. Here, we present an alternative strategy in which the glmS ribozyme is inserted in the C‐terminal region of one allele of a GOI and effectively knocks it down in response to the presence of glucosamine in the culture medium. Using several endogenous genes, we show that the glmS ribozyme cleaves the mRNA in vivo leading to reduction in mRNA and protein expression following glucosamine treatment in both T. brucei procyclic and bloodstream forms. Glucosamine‐induced ribozyme activation can be rapidly reversed by removing the inducer. In summary, the glmS ribozyme could be used as a tool to study essential genes in T. brucei. 相似文献
18.
19.
20.