在单核级LINE-1逆转录转座子分析
Summary
在这里，我们采用FISH方法在单核级跟踪LINE-1逆转录转座子在肝癌细胞系稳定表达合成LINE-1的染色体价差 。
Abstract
Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5′ truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.
Introduction
人长散布核元件-1（ 行1或L1）是通过基因组内传播“复制和粘贴”反转录机制的自主移动元素。一个典型的人类L1是〜6 kb的，由充当启动子的5'UTR（非翻译区）的，两个开放阅读框（ORF）：L1 -ORF1和L1 -ORf2，并与聚腺苷酸一个3'UTR 。尾巴L1 -ORF1蛋白质具有三个不同的区域：一个线圈螺旋结构域，RNA识别基序和C末端结构域，而L1 -ORf2蛋白质具有核酸内切酶，逆转录酶和富含半胱氨酸的结构域1,2,3,4,5。 L1 -ORF1表现出的核酸分子伴侣的活动，而L1 -ORf2提供酶活性，与逆转录转座子1,2,6所需的两种蛋白。
L1逆转录转座子的周期从L1的5'UTR转录启动</EM>由RNA聚合酶II，易位进入细胞质和翻译表达双顺反子。在细胞质中，L1 -ORF1展品无论是顺式结合位它打包了自己的RNA或反式结合位它打包其他的RNA（ 即 SINE / SVAS /假）与L1 -ORF2p 7形成核糖核蛋白颗粒（RNP） 8。的RNP易位进入细胞核其中L1 -ORF2p刻痕基因组DNA（gDNA的）的核酸内切酶活以暴露的OH –基团9，即轮流使用逆转录酶素和反向合成的RNA，从3'末端的DNA。期间反转的合成，DNA的第二链被缺口从原始切口部位7-20碱基创建交错符它们填充以形成签名L1插入序列（ 例如 ，TTTTAA）称为目标部位重复（TSD）9,10。这个过程被称为目标素逆转录（TPRT）并导致inserti上在插入序列的两端的L1 /与TSDS其他DNA的完整或截短的副本。L1反转录也已经显示，通过介导TPRT状非同源末端接合在一些细胞类型11。
在我们的研究中所用的L 1反转录载体是非附加型和由标签L1 -ORF1＆2P通过组合CMV-L1-5'UTR启动子驱动（ 图1）12的。这种结构的早期版本已在使用酵母和人类细胞培养13,14,15研究进行了描述。位于5'和3'两个不同的CMV启动子的载体的结束时，用3'末端放置在反向方向，剪接和融合后带动新霉素的表达。在3'末端的反转录指示符盒由一个新霉素基因插入的反义到L1 -ORFs的和的glob呈现由分离非活性成两半在与拼接供体（SD）和剪接受体（SA）位点（ 图1）的内含子。一旦整合到染色体中，L1是从共同的启动子转录产生，它由一个双顺反子mRNA的和不活动的新霉素基因的mRNA。期间RNA加工，球蛋白内含子剪接出来的新霉素基因的恢复一个全功能的新霉素基因。该混合的mRNA包装成在顺一个RNP和易位进入它被整合到基因组作为使用TPRT任全长或截短的插入的核。
在这里，我们描述了使用荧光原位杂交（FISH）用专门针对拼接新霉素基因（SNeo）探针在单核水平来跟踪L1 -retrotransposiition型态和插入率的方法。效率和检测的特异性是使用反转录称职和不称职的结构和探针DET证实等SNeo或新霉素和珠蛋白内含子交界处16。这种方法占一些细胞培养基于反转录测定法，如多次插入，菌落电阻和有利的克隆扩充的缺点。
Protocol
Representative Results
Discussion
方法，如全基因组测序，反向PCR和Southern杂交已用于研究L1逆转录转座子。虽然这些方法是在基因组内发生L1插入定位极有价值，为所有这些混杂的挑战是需要在硅片编程进行重组序列。这里所描述的FISH方法被设计以补充这些方法，特别是在需要在培养细胞中的异位L1反转录的分析研究的情况。该方法可用于反转录事件的定量和定性评价和施加到间期核。这种方法拥有的?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
This work was supported in part by grants from the National Institute of Environmental Health Sciences (ES014443 and ES017274) and AstraZeneca to KSR.
Materials
| Labeling Probes | |||
| Go Tag DNA polymerase | Promega | M3178 | |
| GO Tag 10X colorless buffer | Promega | M3178 | |
| Individual dNTP | Sigma | DNTP10 | Adjust the concentration of dATP, dGTP, dCTP to 2mM and dTTP to 1.5mM in nuclease free water. |
| dUTP-16-Biotin (0.5mM) | Roche | 11093070910 | |
| dUTP-FITC (0.5mM) | Thermo Scientific | R0101 | |
| dUTP-CY3 (0.5mM) | Sigma | GEPA53022 | |
| MIRUS FISH Labeling Kit | MIRUS | MIR3625 | |
| MIRUS FISH Labeling Kit | MIRUS | MIR3225 | |
| NucleoSpin PCR Clean-Up kit | Qiagen | 1410/0030 | Any PCR clean reagent can be used in place of NucleoSpin PCR Clean Up Kit |
| PCR Machine | Any Thermocycler machine can be used to amplify the label product. | ||
| Agarose | Sigma | A9539 | |
| Name | Company | Catalog Number | Comments |
| Preparing chromosome Spreads. | |||
| Colcemid | Life Technologies | 15212-012 | Add Colcemid directly to growth media to a final concentration of 0.4 µg/mL |
| 1M KCl | Sigma | P9541 | Dilute 1M KCl solution to 75 mM solution to make Hypotonic Solution |
| Carnoy Fixative Solution | Mix 3 volumes of methanol and 1 volume of acetic acid to make Carnoy Fixative Solution. Make Fresh everytime. | ||
| Methanol | Sigma | 322415 | |
| Acetic acid | Sigma | 320099 | |
| Hoechst-33342 | Thermo Scientific | 62249 | Dissolve Hoechst-33342 in PBS to final concentration of 1 mg/mL. |
| Diamond Point Marker | Thermo Scientific | 750 | |
| Frosted Slides | Thermo Scientific | 2951-001 | |
| Trypsin-EDTA (0.25%) | Life Technologies | R001100 | To detech cells, incubate cell in Trypsin for 5 mins and inactive with equal volume of complete media |
| Cover slides | VWR | 48366067 | |
| Coplin Jars | Thermo Scientific | 107 | |
| Heat Block | Any heat block can be used for this purpose, though blocks fitted for heating slides are recommended. | ||
| Beaker (600ml) | Sigma | CLS1003600 | Fill the beaker with water, heat to boil, use the hot steam to burst chromosomes. |
| Nikon Microscope | Nikon | 125690 | Any microscope can be used to look at spread quality. |
| Name | Company | Catalog Number | Comments |
| Reagents and Materials for FISH | |||
| Trisodium citrate | Sigma | S1804 | |
| Sodium Chroride | Sigma | S3014 | |
| Formamide | Sigma | F9037 | |
| Tween-20 | Sigma | F1379 | |
| Detran Sulfate | Sigma | D8906 | |
| SDS | Sigma | L3771 | |
| Salmon Sperm DNA | Life Technologies | 15632-011 | |
| Bovine Serum Albumin (BSA) | Sigma | A8531 | |
| HCl (N) | Sigma | 38283 | |
| Ethyl Alcohol | Sigma | 459844 | |
| Methanol | Sigma | 322415 | |
| DPBS | Life Technologies | 14190-250 | |
| NaOH | Sigma | S8045 | |
| Rnase A | Sigma | R4642 | |
| Pepsin | Sigma | P6887 | |
| Paraformaldehyde (PFA) | Sigma | P6148 | |
| Hoechst-33342 | Thermo Scientific | 62249 | Dissolve Hoechst-33342 in PBS to final concentration of 1 mg/mL. |
| Rubber Cement/Cytobond Sealant | 2020-00-1 | ||
| Seven Coplin Jars | Thermo Scientific | 107 | |
| Dark Humidified chamber | Sigma | CLS2551 | |
| Cover slides | VWR | 48366067 | |
| Dry Digital Heat Block | VWR | 13259 | |
| Fluorescence Microscope | |||
| 20X Saline-Sodium Citrate (20X SSC) | Combine 175 g of NaCl, and 88.2 g of trisodium citrate with 800 mL of molecular grade H2O. Stir while adjusting to pH 7. Once the all salts dissolve, adjust the volume to 1 L and filter through 0.22 µm Filter paper. Store at 4 °C. | ||
| 1mg/mL of Rnase A | Dilute 20X SSC buffer to 2X SSC buffer. Dissolve RNase A in 2X SSC buffer to a final concentration of 1 mg/mL. Make fresh solution every time. | ||
| 1% Pepsin | Dissolve pepsin (W/V) in 10 mM HCl to a final concentration of 1 % solution. Make fresh every time. | ||
| 4% Paraformaldehyde (4% PFA) | Weigh 4.0 g of PFA in fume hood, add 50 mL of 1xPBS, heat to 60 °C while stirring and adjust the pH with drops of NaOH until all PFA dissolves and the solution becomes clear. Adjust the volume to 100 mL, filter through 0.22 µm Filter paper and store 5 mL aliquots at -20 °C. Thaw aliquot of PFA at 37 °C for 10-15 min for subsequent uses. Adjust filter size depending on amount of paraformaldehyde. | ||
| Wash Buffer | Combine 100 mL of Formamide (20%) with 2.5 mL of 20X SSC, adjust to pH 7 with 1.0 M HCl and bring the volume to 500 mL with molecular grade H2O. | ||
| Hybridization Buffer | Combine 50% formamide, 10% dextran sulfate, 0.1% SDS, and 300 ng/mL Salmon Sperm DNA in 2X SSC buffer. Amount of Formamide determine how focused chromatids are, titrate the amount | ||
| Detection Buffer | Dilute 20X SSC buffer to 4X and add 0.2% Tween-20 to make detection buffer. | ||
| Blocking Buffer | Combine 5% bovine serum albumin (BSA) with 0.2% Tween-20 in 4X SSC buffer. | ||
| Dehydrating Solution | Make 70%, 80%, and 95% ethanol solutions. |
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