Identification and Monitoring of Nucleotide Repeat Expansions Using Southern Blotting in Drosophila Models of C9orf72 Motor Neuron Disease and Frontotemporal Dementia
应用Southern Blotting鉴定和监测C9orf72运动神经元疾病和额颞叶痴呆模型的核苷酸重复扩增
发布: 2022年05月20日第12卷第10期 DOI: 10.21769/BioProtoc.4424 浏览次数: 2567
评审: Oneil Girish BhalalaThomas MoensSébastien Gillotin
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The authors used this protocol in:
Sep 2020
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Abstract
Repeat expansion diseases, including fragile X syndrome, Huntington’s disease, and C9orf72-related motor neuron disease and frontotemporal dementia, are a group of disorders associated with polymorphic expansions of tandem repeat nucleotide sequences. These expansions are highly repetitive and often hundreds to thousands of repeats in length, making accurate identification and determination of repeat length via PCR or sequencing challenging. Here we describe a protocol for monitoring repeat length in Drosophila models carrying 1,000 repeat C9orf72-related dipeptide repeat transgenes using Southern blotting. This protocol has been used regularly to check the length of these lines for over 100 generations with robust and repeatable results and can be implemented for monitoring any repeat expansion in Drosophila.
Keywords: Southern blotting (DNA印迹术)
Drosophila (果蝇)
C9orf72 (C9orf72)
Dipeptide repeats (二肽重复)
Repeat expansions (重复扩展)
Background
Repeat expansion diseases are a class of genetic disorders associated with the expansion of tandem repeat DNA sequences. The polymorphic nature and inherent instability of tandem repeats renders them prone to mutation, and as such they are one of the most abundant causes of variation in the human genome (Gymrek, 2017). The length and location of repeat sequences can vary, ranging from a single nucleotide to several nucleotides per repeat, situated within both coding and non-coding regions. There have been over 50 repeat expansion disorders identified, most of which primarily affect the central nervous system (Depienne and Mandel, 2021).
Repeat expansions in coding regions tend to be trinucleotide repeats, resulting in abnormally long repetitive amino acid sequences within proteins. In contrast, expansions in the UTRs or introns of genes are more varied in terms of sequence and how they confer toxicity. The repeat sequence itself can vary in length, the longest reported being a dodecamer in the 5’UTR of the CSTB (cystatin B) gene, which results in progressive myoclonic epilepsy type 1 (EPM1) (Lalioti et al., 1997). Moreover, whilst GC-rich repeat expansions in the 5’UTR tend to cause loss of function toxicity via epigenetic mechanisms such as persistent DNA hypermethylation, expansions situated within 3’UTRs and introns more frequently lead to a gain of function toxicity through RNA toxicity or polypeptide synthesis via repeat associated non-AUG (RAN) translation. An example of a repeat expansion that both reduces gene expression and elicits RNA and polypeptide repeat toxicity is the intronic GGGGCC expansion in the C9orf72 gene, the leading genetic cause of frontotemporal dementia (FTD) and motor neuron disease (MND) (Renton et al., 2011; DeJesus-Hernandez et al., 2011). Research using genetic models, including fruit flies, has determined that, whilst the C9orf72 expansion does reduce gene expression and cause haploinsufficiency, the main driver of toxicity in this case is the production of dipeptide repeat proteins (DPRs) from non-canonical RAN translation of the repeat itself. As translation occurs in all frames and from both sense and antisense RNA, five different DPRs are produced: glycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR), alanine-proline (AP), and proline-arginine (PR) (Mori et al., 2013). These DPRs have been shown to aggregate in patient brains and spinal cord and are toxic in multiple model systems (May et al., 2014; Mizielinska et al., 2014; Zhang et al., 2014; Moens et al., 2019; West et al., 2020).
To further our understanding of expansion disorders and the mechanisms underpinning them, a range of transgenic model systems have been developed. For each expansion disorder, there is usually a consensus as to what constitutes a pathogenic repeat length. For example, the intronic hexanucleotide GGGGCC repeat in the C9orf72 gene is normally under 30 repeats in healthy individuals, whereas in those who develop disease it comprises over 500 and often thousands of repeats (Renton et al., 2011; DeJesus-Hernandez et al., 2011). In West et al. (2020), we developed the first Drosophila models expressing each DPR, individually and without repeat RNA, at over 1,000 repeats. To do this, alternative coding sequences were designed to produce the DPRs independently of the GGGGCC repeat (Callister et al., 2016). However, the transgene is still highly repetitive, GC-rich, and over 6000 bp in length, making accurate genotyping of repeat length via PCR or sequencing challenging. Furthermore, tandem repeats are increasingly unstable in a length dependent manner (Depienne and Mandel, 2021), and repetitive sequences introduced into bacteria and animal models are known to retract or be excised completely through generations (Bichara et al., 2006; Ryan et al., 2019). Therefore, it is important to be able to determine the length of repeat sequences in transgenic models and monitor them to check for retractions. The most common method, and widely considered gold standard, is Southern blotting.
Southern blotting is a highly sensitive technique used to detect specific DNA sequences in a blood or tissue sample. It involves the digestion of DNA with site-specific restriction endonucleases to isolate the genomic region of interest, followed by the separation of DNA fragments by size using gel electrophoresis and transfer onto a porous, positively charged nylon or nitrocellulose membrane via capillary action. The DNA of interest is detected using molecular hybridisation with specific nucleic acid probes, which are tagged with either radioisotopes or non-isotopic reagents (e.g., to facilitate chemiluminescent detection) (Southern, 2006). Whilst Southern blotting has been largely replaced by modern sequencing techniques and is no longer widely used, it remains an essential technique for researchers working with long and repetitive DNA sequences. Repeat sequences have always proved technically challenging for sequence alignment and assembly, and this is exacerbated when the repeat exceeds the sequencing fragment length (typically 350–500 bp). Moreover, traditional PCR amplification libraries have an inherent GC-bias, which can result in under-representation of reads from repeat expansions, a large proportion of which have 100% GC content (Treangen and Salzberg, 2012). These inaccuracies can lead to under-estimation of repeat sizes and mischaracterisation of repeat expansions (Rajan-Babu et al., 2021). It has been reported that PCR-based techniques used to detect the C9orf72 expansion in FTD/MND patients are unreliable, with both a high false-positive and false-negative rate (Akimoto et al., 2014). Therefore, whilst there are promising developments in long-read sequencing analysis, at present Southern blotting is considered the gold standard for genotyping GC-rich repeat expansions. Here we have developed and refined a protocol for the Southern blotting of repeat sequences in Drosophila. We optimized this protocol using Drosophila C9orf72 DPR models published in West et al. (2020), but it can be applied to other repeat expansions with alterations to the procedure required only for the oligonucleotide probes and restriction endonucleases.
Materials and Reagents
Whatman® 3MM filter paper (Whatman®, GE Healthcare, catalog number: WHA30306185)
Extra thick blotting paper (2.5 mm) (ThermoFisher Scientific, Thermo ScientificTM, catalog number 88605)
AmershamTM HybondTM-N 0.45 µm pore, neutral nylon membrane (GE Amersham, catalog number: RPN203N)
15 mL Falcon tubes (e.g., Corning® 15 mL centrifuge tubes, Sigma-Aldrich, Merck, catalog number: CLS430790)
50 mL Falcon tubes (e.g., Corning® 50 mL centrifuge tubes, Sigma-Aldrich, Merck, catalog number: CLS430829)
1.5 mL microfuge tubes (e.g., Eppendorf® Safe-Lock microcentrifuge tubes, Sigma-Aldrich, Merck, catalog number: T9661)
Plastic film (e.g., clingfilm) (Scientific Laboratory Supplies, catalog number: FIL1003)
Acetate sheets (Rapid Electronics Ltd, Diacel, catalog number: 34-0303)
Petri dish or weighing boat (for picking heads as shown in Figure 1, e.g., small square weighing boat, Sigma-Aldrich, Merck, catalog number: Z708542-500EA)
Phenol-chloroform-isoamylalcohol (25:24:1) saturated with 10 mM Tris, pH 8.0, 1 mM EDTA (Sigma-Aldrich, Merck, catalog number: P3803)
Chloroform (analytical reagent grade) (Sigma-Aldrich, Merck, catalog number: 366927)
Ethanol (for molecular biology) (Sigma-Aldrich, Merck, catalog number: 51976)
Ethidium Bromide, Molecular Biology-grade Aqueous Solution (500 mg/mL) (Sigma-Aldrich, Merck, catalog number: E1385)
Concentrated (36.5–38.0%) hydrochloric acid (HCl) (Sigma-Aldrich, Merck, catalog number: H1758)
Sodium chloride (NaCl) (analytical reagent grade) (Sigma-Aldrich, Merck, catalog number: S9888)
Sodium hydroxide (NaOH) pellets (anhydrous) (Sigma-Aldrich, Merck, catalog number: S5881)
Sodium citrate (Sodium citrate tribasic dihydrate) (Sigma-Aldrich, Merck, catalog number: C8532)
TRIS hydrochloride (TRIS HCl) pH 8.0 (Sigma-Aldrich, Merck, catalog number: 10812846001)
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, Merck, catalog number: L3771)
Ethylenediaminetetraacetic acid (EDTA) (Merck, Sigma-Aldrich, catalog number: E9884)
Drosophila stocks to be tested (50–60 flies per genotype (see Notes for applying this to other tissues/protocols), including a negative wild-type control such as Canton S or Oregon R). Wild-type flies can be acquired from Bloomington Drosophila Stock Centre (BDSC, Indiana University) (Canton S: BDSC 9515, Oregon R BDSC 2376) or other stock centre. Transgenic stocks used in our example were generated by microinjection of pUAST-DPR-EGFP into VK00005 embryos by Cambridge Microinjection Facility [see West et al. (2020); available upon request].
Plasmids used to generate the transgenic fly lines, for use as positive controls [e.g., pUAST-DPR(1000)], see West et al., 2020, available upon request
Dry ice
Nuclease free water (molecular biology grade) (Sigma-Aldrich, Merck, catalog number: W4502)
Proteinase K (ThermoFisher Scientific, Thermo ScientificTM, catalog number: EO0491), divide into 1 mL aliquots and store at -20°C
Restriction enzymes to excise DNA of interest from the genome with appropriate buffer; for our example, we used Dde1 (New England Biolabs, catalog number: R0175) and NlaIII (New England Biolabs, catalog number: R0125), along with CutSmart® buffer (New England Biolabs, catalog number: B7204).
TBE Buffer (Tris-borate-EDTA) (10×) (ThermoFisher Scientific, Thermo ScientificTM, catalog number: B52)
Agarose for molecular biology (Sigma-Aldrich, Merck, catalog number: A9539)
6× loading dye containing bromophenol blue (New England Biolabs, catalog number: B7021S)
DNA Molecular Weight Marker II, DIG-labeled (Roche, Merck, catalog number: 11218590910)
1 kb Plus DNA Ladder (NEB, catalog number: N3200)
Probes specific to target sequence [The probes used in West et al., (2020), synthesised by Eurofins UK, are listed in the notes section of this protocol].
DIG easy hyb (Roche, Merck, catalog number: 11603558001)
Salmon sperm DNA (Agilent Technologies, catalog number: 201190), divide into aliquots of 300 µL and 150 µL and store at -20°C.
DIG Wash and Block Buffer Set (Roche, Merck, catalog number: 15857 62001), aliquot DIG block (e.g., 50 mL aliquots) and store at -20°C for long term (one aliquot can be stored for up to 3 months at 4°C).
Anti-Digoxigenin-AP, Fab fragments (Roche, Merck, catalog number: 11093274910, RRID:AB_2734716), store at 4°C
Ultrapure water (for example from Milli-Q® Purification System)
CDP-Star® chemiluminescent substrate (Roche, Merck, catalog number: CAS160081-62-9), store at 4°C
70% Ethanol (see Recipes)
Depurination solution (see Recipes)
Gel denaturing solution (see Recipes)
Gel neutralizing solution (see Recipes)
20× SSC stock (see Recipes)
10% SDS Solution (see Recipes)
2× SSC, 0.1% SDS (see Recipes)
0.5× SSC, 0.1% SDS (see Recipes)
0.1× SSC, 0.1% SDS (see Recipes)
Positive controls (see Recipes)
Genomic extraction buffer (see Recipes)
1× TBE (see Recipes)
Ladder mix (see Recipes)
1× DIG block (see Recipes)
1× maleic acid buffer (see Recipes)
1× maleic acid wash buffer (see Recipes)
1× detection buffer (see Recipes)
Equipment
Pipettes and tips (P1000, P200, P20, P10, and P2)
Fine paintbrush (e.g., RS PRO Thin 6.4 mm paintbrush, RS Components Ltd, catalog number: 2379190)
Vortex mixer (e.g., Vortex-Genie® 2 mixer, Sigma-Aldrich, Merck, catalog number: Z258415)
Fume hood
pH meter
Standard 700W–1000W microwave
Standard laboratory microfuge (e.g., Sigma 1-14 Microfuge, Sciquip, catalog number: 90616)
Tabletop mini microfuge (e.g., SciSpin MINI Microfuge, Sciquip, catalog number: SS-6050)
Wheel tube rotator (e.g., Cole-ParmerTM Stuart TM Rotator Disk, Fisher Scientific, catalog number: 11496548)
PowerPacTM Basic Power Supply (Bio-Rad, catalog number: 1645050)
Sub-Cell GT Electrophoresis Cell with 20-well combs and gel casting tray (15 × 15 cm) (Bio-Rad, catalog number: 1704402)
Clean plastic trays and sandwich boxes for incubating the membrane and gel, and assembling the Southern blot (Figure 2)
Large sandwich container (e.g., 250 × 150 mm) to use as the basin for Southern blotting apparatus (Figure 2)
Chemiluminescence and fluorescence imaging system (e.g., G:box imaging unit, Syngene)
HB pencil
Scissors/guillotine for cutting filter paper and membrane
UV Transilluminator (e.g., dual wavelength (302/365) 8W transilluminator LM-20, VWR, catalog number: 732-4388)
Standard boiling water bath (e.g., VWR, SBB Aqua 5 plus, catalog number: 462-0171) or hot block capable of reaching >100°C
Standard hybridisation oven (e.g., HB-1000 Hybridiser, VWR, catalog number: 732-4300)
Glass hybridisation bottles to fit in hybridisation oven (e.g., 35 × 150 mm hybridisation bottles, VWR, catalog number: 732-4350)
Flat edged forceps (e.g., S MurrayTM Stainless Steel Forceps L325/01, Fisher Scientific, catalog number: 12342158)
Rocker (e.g., Cole-ParmerTM StuartTM See-Saw Rocker, catalog number: 10470655)
Software
Imaging software for imaging the blot, for example: GeneSys and GeneTools (Syngene, https://www.syngene.com/support/software-downloads/)
Procedure
文章信息
版权信息
© 2022 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Sharpe, J. L., Harper, N. S. and West, R. J. H. (2022). Identification and Monitoring of Nucleotide Repeat Expansions Using Southern Blotting in Drosophila Models of C9orf72 Motor Neuron Disease and Frontotemporal Dementia. Bio-protocol 12(10): e4424. DOI: 10.21769/BioProtoc.4424.
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分类
神经科学 > 神经系统疾病 > 神经退行性病变
神经科学 > 神经系统疾病 > 动物模型
生物化学 > DNA
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