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Dec 2018
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A High-throughput qPCR-based Method to Genotype the SOD1G93A Mouse Model for Relative Copy Number
一种基于qPCR 的高通量研究 SOD1G93A小鼠模型中相对拷贝数的基因分型方法   

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Abstract

The most commonly used mouse model in ALS preclinical research expresses multiple copies of the human SOD1 (G93A) transgene. During the course of breeding, successive generations of mice can lose copies of the transgene. Because shorter lifespan of these mice is dependent on transgene copy number, it is essential to ensure that no low-copy, and therefore longer-lived, mice are included in preclinical studies. Existing techniques for SOD1G93A mouse genotyping are broadly based on creating a standard curve using a reference gene and deducing the relative amount of SOD1 by comparison with the standard curve. This type of technique is used in Alexander et al. (2004), Vieira et al. (2017) and Maier et al. (2018). However, it is not described in detail (see Note 1). This paper provides a detailed protocol for determining the relative copy number of the human SOD1 transgene. Briefly, the protocol involves first the extraction of high-quality genomic DNA from mouse ear tissue, creation of a genomic DNA concentration-based standard curve, and qPCR analysis of up to 88 samples at once alongside the standard curve with Gapdh as a reference gene. Analysis involves the normalization of each unknown sample using the standard curve followed by determination of the copy number of the sample relative to the cohort median. This protocol has been optimized to produce high-quality genomic DNA and consistent results, and the relative copy number cutoffs have been optimized and validated empirically by comparison of relative copy number and mouse lifespan.

Keywords: ALS mouse model (ALS小鼠模型), Mouse genotyping (小鼠基因型), SOD1 (SOD1), Transgene copy number (转基因拷贝数目), Preclinical research (临床前研究), High-throughput genotyping (高通量基因分型), Relative copy number (相对拷贝数)

Background

The SOD1G93A mouse model (B6SJL-Tg(SOD1*G93A)1Gur/J) is currently the most commonly used mouse model for preclinical testing of therapies for amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease with most patients living between two and five years after diagnosis (Morrice et al., 2018). The human SOD1 transgene is present in multiple copies and mouse lifespan inversely correlates with copy number. Alexander et al. (2004) found that mice with 24 copies of the SOD1(G93A) gene lived to approximately 129 days, whereas mice with 34 copies lived to 99 days and mice with 20 copies lived to 150 days (Alexander et al., 2004). Additionally, 3.6% of high-copy transgenic male breeders had offspring with low copy numbers over the course of four years, signifying that it is not enough to only test stud males for copy number (Alexander et al., 2004). Although higher copy does correlate with shorter lifespan, spontaneous recombination tends toward dropped copies rather than increased copy number (Alexander et al., 2004; Zwiegers et al., 2014). Therefore, we focused our assay on identifying low-copy mice only.

In order to be sure that any measurable effects in a preclinical drug study are attributable to the treatment and not to an inherent difference in transgene copy, it is absolutely essential that no low-copy mice are included. While there are no SOD1 (G93A) genotyping methods published in a dedicated method or protocol journal, there are papers which briefly describe genotyping in the methods section. For example, Alexander et al. (2004) describe a qPCR-based method that uses change in cycle threshold (∆Ct) of SOD1 (G93A) and a reference gene to quantify copy number difference (Alexander et al., 2004).

In our hands, we found that published genotyping methods prior to 2016 were not sensitive enough to exclude all low-copy animals. Figure 1 shows the age at death of all mice included in a non-treatment group of survival studies from August 2015 to January 2019. The x-axis is the start date of each study, and the data set for each start date is the death ages of the mice included in the untreated group of that study. The finalized genotyping protocol detailed in this paper was instated on May 9, 2016, and the first mouse genotyped with this protocol was included in the study with the start date of June 7, 2016. The graph was analyzed in GraphPad Prism using a Box-and-Whiskers Tukey Test, which highlights any value that is either greater than the 75th percentile plus 1.5 times the interquartile range (IQR) or less than the 25th percentile minus 1.5 times the IQR. These outliers are shown as large blue dots. Since the genotyping protocol was instated, the number of outliers and their distance away from the median tend to decrease.


Figure 1. GraphPad Prism analysis of mouse death ages from August 2015 to January 2019. These mice were in untreated groups of survival studies. The dates shown on the x-axis indicate the start date of each study, and the data set associated with each date represents death ages of the mice in the untreated group of each study. Tukey’s Test on box-and-whisker plots has been applied, with the blue dots showing which data points are outside the 75th percentile plus 1.5 times the interquartile range (IQR) or the 25th percentile minus 1.5 times the IQR.

This genotyping protocol has been used in several published studies since 2016, including Vieira et al. (2017), and provides a qPCR-based method to ascertain relative copy number in a cohort of up to 88 mouse ear tissue samples without the need to determine absolute gene copy. The protocol relies on a concentration-based Gapdh standard curve to normalize the samples. After normalization, relative copy numbers are assigned based on log difference between each sample and the cohort median. Along with developing a reliable method to screen for low-copy mice, we optimized tissue collection to avoid cross-contamination and optimized genomic DNA extraction for consistent yield and high quality (see Note 2). Additionally, we also performed a dilution qPCR of potential reference gene primer and probe sets to determine which correlated best with the custom SOD1 target primer/probe set, and to determine the concentration range of our standard curve (Figure 2). As shown with the green triangles, the copy-number Gapdh primer/probe set from Thermo Fisher had the most comparable slope and Ct values to the SOD1 primer/probe sets, and both performed best between 2.5 and 0.07 ng/μl genomic DNA. To determine the safest relative copy number cutoff, we performed a survival study correlating copy number with lifespan. Mice with 16 or more copies showed typical lifespans for this mouse model, while mice with 15 or fewer copies were long-lived (Figure 3). Using this information we determined the safest cutoff to be 15 copies and fewer.


Figure 2. A qPCR experiment performed with serial two-fold dilutions of SOD1G93A genomic DNA from mouse ear tissue, probed with our custom SOD1 primer/probe set as well as several off-the-shelf primer/probe assays from Thermo Fisher. Our custom SOD1 primer/probe set compares well with the off-the-shelf SOD1 assay, and the Gapdh reference assay is closest to the SOD1 target assays in both Ct and in slope of the linear region. The graph is most linear between 0.07 and 2.5 ng/μl of genomic DNA, which gave us the optimal range for our experimental standard curve.


Figure 3. This graph shows the survival of SOD1G93A mice compared to their relative copy number as assigned by the genotyping protocol. The mice within a tolerable range of death ages were those in the 16-23+ copy number groups. Therefore, we selected 15 as the cutoff for relative copy number.

Materials and Reagents

Note: All storage is at room temperature unless otherwise specified.

  1. QIAcube HT Plasticware (QIAGEN, catalog number: 950067)
  2. Adhesive PCR Plate Seals (Thermo Fisher, Thermo Scientific, catalog number: AB0558)
  3. Sterile Alcohol Prep Pads (Fisher Scientific, Fisherbrand, catalog number: 22-363-750)
  4. 15 ml Centrifuge Tube (Corning, catalog number: 430766)
  5. 96-well UV-Transparent Microplates (Corning, catalog number: 3635)
  6. 384-well qPCR plate (Thermo Fisher, Applied BioSystems, catalog number: 4309849) 
  7. Custom SOD1 MGB probe and primers (Thermo Fisher, Custom Genomics, -20 °C)
    1. Probe: SOD1HumanV2-55T (VIC-ACTCTCTCCAACTTTG) with VIC fluorescent reporter chemistry, 6000 pmole (wet, desalt) 
    2. Forward primer: SOD1HumanV2-27F (GTAAATCAGCTGTTTTCTTTGTTCAGA), 80,000 pmole (dry, desalt)
    3. Reverse primer: SOD1HumanV2-104R (TTCACTGGTCCATTACTTTCCTTTAA), 80,000 pmole (dry, desalt)
  8. QIAamp 96 QIAcube HT Kit (QIAGEN, catalog number: 51331)
  9. PBS, Phosphate Buffered Saline, 10x Solution (Fisher BioReagents, Fisher Scientific, catalog number: BP3994)
  10. Alcohol, Reagent (Denatured Alcohol), 70% (v/v) Aqueous Solution (Ricca Chemical Company, Fisher Scientific, catalog number: 25467032, room temperature flammables cabinet)
  11. Neosporin First Aid Antibiotic Ointment (Hanna’s Pharm Supply Co, catalog number: 302-571-8761)
  12. Cotton-Tipped Applicators (Fisher Scientific, Puritan, catalog number: 22-029-571)
  13. 50 ml Reagent Reservoir (Corning, Costar, catalog number: 4870)
  14. DL-Dithiothreitol solution BioUltra for molecular biology, 1 M in H2O (Millipore Sigma, Sigma-Aldrich, catalog number: 43816), 4 °C
  15. Ethanol, Absolute (200 Proof), Molecular Biology Grade (Fisher Scientific, catalog number: BP2818100), room temperature flammables cabinet
  16. Nuclease-Free Water (Thermo Fisher, Invitrogen, catalog number: AM9932)
  17. TaqMan Genotyping Master Mix (Thermo Fisher, Applied Biosystems, catalog number: 4371355), 4 °C
  18. TaqMan Gapdh Copy Number Assay (Thermo Fisher Assay ID Mm00186822cn), -20 °C
  19. Sample Prep solution (see Recipes)
  20. Tissue Digestion solution (see Recipes)
  21. Primer Dilution solution (see Recipes)
  22. SOD1G93A Copy Number Genotyping qPCR Master Mix (see Recipes)
  23. Primer/Probe solution (see Recipes)

Equipment

  1. Two sterilized vessels for tissue harvest ethanol washes
  2. Surgery-quality tools to harvest ear tissue (forceps and scissors, or ear punch)
  3. Shaker capable of 18 h of 200 rpm and 56 °C incubation (Eppendorf, New Brunswick, catalog number: M1352-0010) (see Note 3)
  4. Qiagen QIAcube HT (QIAGEN, catalog number: 9001793) (see Note 3)
  5. SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices, catalog number: M5) (see Note 3)
  6. NanoDropTM 8000 Spectrophotometer (Thermo Fisher, Thermo Scientific, catalog number: ND-8000-GL) (see Note 3)
  7. Allegra X-22R Benchtop Centrifuge (Beckman Coulter, catalog number 392188) (see Note 3)
  8. QuantStudioTM 7 Flex Real-Time PCR System, 384-well, desktop (Thermo Fisher, catalog number: 4485701) (see Note 3)

Software

  1. Excel 2013 (Microsoft)
  2. Prism 6 (GraphPad, www.graphpad.com)

Procedure

  1. Tissue collection
    Note: Although this protocol specifies extracting and analyzing genomic DNA from ear samples, 20 mg mouse tail tip samples may also be used.
    1. Preparation
      1. Create a plate map with samples organized top to bottom in columns (i.e., Sample 1 is in spot A1, Sample 2 is in spot B1, etc.). Remember that in every experiment, 8 sample spots need to be reserved for a standard curve, so the most mice that can be analyzed in a single qPCR is 88 (Figure 4A).
      2. Create Sample Prep solution (Recipe 1) with appropriate excess (at least 10). 
      3. Using an S-Block from the Qiagen QIAcube Plasticware kit, load 160 μl of the Sample Prep solution into every well of every column needed for the experiment (Figure 4B).
        Note: The QIAcube HT works in column format, and every well of the columns that are used (regardless of whether they contain a sample) needs to be filled in order for the automated protocol to work properly.
      4. Seal the top of the S-block with an adhesive plate seal.


        Figure 4. Sample collection plate example. A. Sample collection plate map for an 88-sample experiment, with placeholder wells for the standard curve. B. Qiagen S-Block.

    2. Sample Collection
      1. Fill two clean vessels with enough 70% reagent alcohol to wash tools.
      2. Pre-clean tools with 70% reagent alcohol.
      3. Collect an ear sample from each mouse that is 20 mg or less using scissors or ear punch (Figure 5); using forceps, place sample into the appropriate well according to the plate map.
      4. Apply antibiotic ointment to mouse ear using cotton-tipped applicator.
        Note: The antibiotic swab does not usually need to be cleaned between uses, but if the mouse bleeds, we replace the soiled swab.
      5. Swish forceps and scissors in first alcohol wash, wipe with alcohol prep pad to remove any solid material such as hairs, and then swish in second alcohol wash. Leave tools in the second wash until ready to use again; do not mix up washes because the first wash should function as a “dirty wash” and the second should function as a “clean wash”.
      6. Repeat with remaining mice, changing the alcohol prep pad and cleaning gloves often.
      7. Cover S-block with adhesive when you’re done and during any extended breaks.


        Figure 5. An ear tissue sample that is approximately 20 mg

    3. Sample digestion
      1. In reagent reservoir, create Tissue Digestion solution (Recipe 2) with appropriate excess (at least 10), mix well, and aseptically load 40 μl into sample wells in S-block; load recipe into any “blank” wells too (wells in a used column that do not have a sample).
      2. Ensure that every sample is submerged.
      3. Seal S-block with a thermally stable adhesive plate cover; add extra security with lab tape so that the adhesive cover does not pop off during overnight digestion (samples will evaporate if this happens).
      4. Secure in a thermal shaker set to 56 °C; shake at 200 rpm overnight or up to 18 h.

  2. Genomic DNA extraction
    1. QIAcube HT setup
      1. After overnight sample digest, open QIAcube HT’s QIAamp 96 protocol.
      2. Follow the setup wizard in the QIAamp 96 QIAcube HT protocol to input sample number and make the following customizations.
        1. Make elution volume 150 μl (Figure 6A).
          Note: In our hands, 150 μl elution produces sample yield between 10-50 ng/μl, which we found to be the best range for sample dilution. You may need to change elution volume to optimize yield range. 
        2. Remove Top Elute (change volume to 0) (Figure 6B).
          Note: With experimentation, we found that using Top Elute is messy and does not improve sample yield or quality.
      3. Set up deck according to the specifications on the software program.
      4. Deionize the deck with an ionizing gun or fan prior to running the protocol.


        Figure 6. QIAcube setup. A. Set Elution Buffer to 150 μl. B. Set Top Elute volume to 0 μl.

    2. Storage: once the protocol is finished, cover the elution plate with adhesive tape and lid, label, and store at 4 °C until ready to use.
      Note: The kit instructs the user to use the rubber tube caps, but we find that to be cumbersome and have an increased chance of liquid flicking into another well.

  3. Genomic DNA Dilution
    1. Use a plate reader or MultiDrop to take 260 nm, 280 nm, and 320 nm readings of the DNA (if using a plate reader, dilute gDNA 1:10 in water in a UV plate).
    2. Quantify the concentration and 260/280 ratio; ensure that 260/280 values are approximately 1.8.
      Note: Another ratio that indicates sample purity is 260/230. Ideal 260/230 values are at least 1.0, but we have not found that 260/230 values have a significant effect on qPCR results so it is not further addressed in this protocol.
    3. Find the samples with the highest concentration; choose one dilution factor for the whole plate that ensures that the highest concentration sample does not exceed 1 ng/μl and the lowest concentration sample is not lower than 0.02 ng/μl.
    4. Dilute all samples in PCR-clean water in a new plate and mix well.

  4. Standard curve setup
    1. Combine and mix several samples’ worth of high-quality genomic DNA made from several different SOD1G93A mice to a total of approximate 100 μl of mixed sample.
    2. Find the concentration (ideally, use a NanoDrop for most accurate results); take a few replicates to make absolutely sure.
    3. Dilute the sample down to 2 ng/μl in 80 μl volume with PCR-clean water; save remaining mixed gDNA stock at -20 °C for future standard curves.
    4. Starting with 70 μl of 2 ng/μl gDNA, make 8 serial two-fold dilutions in PCR-clean water in one column of a PCR plate; this creates a standard curve where the most-concentrated sample is 2 ng/μl and the least is 0.016 ng/μl (Figure 7).
      Note: You can make more than one standard curve at a time if you use them a lot, and store them at 4 °C.
    5. Cover with adhesive plate sealer and store at 4 °C. Use aseptic technique every time you use the standard curve.


      Figure 7. Standard curve setup in a 96-well mixer plate

  5. qPCR setup
    1. Primer setup
      1. Dilute dry primers according to the “Primer Dilution solution” (Recipe 3); leave on the bench for 10 min, vortex, and spin down.
      2. Aliquot into 100 μl aliquots, label, and store at -20 °C.
      3. Aliquot wet probes into 20 μl aliquots, label, and store at -20 °C.
    2. qPCR Master Mix
      1. Make master mix according to the “SOD1G93A Copy Number Genotyping qPCR Master Mix solution” (Recipe 4), using appropriate excess (be sure to include standard curve in sample number calculations and make enough for two genes, SOD1 and Gapdh).
      2. Create SOD1 and Gapdh mixes according to the “Primer/Probe solution” (Recipe 5), using appropriate excess. Remember when calculating excess that you can use at most half of the number of excess samples that you used for the master mix for the SOD1 and Gapdh mixes. Mix gently; store at 4 °C covered with foil until ready to use (not more than 1-2 h).
      3. Label 2 96-well PCR plates “SOD1” and “Gapdh”, respectively.
      4. Aliquot 4 μl of diluted unknown gDNA into each plate.
      5. Add 4 μl of standard curve after the last column of samples on the Gapdh plate.
      6. Aliquot 20 μl of SOD1 master mix to each sample well of the SOD1 plate; pipette mix very well but do not introduce bubbles. Repeat with Gapdh plate and master mix.
      7. Spin plates at 180 x g for 3 min.
      8. Starting with the SOD1 plate, pipette 9.8 μl duplicates across a 384-well qPCR plate with an 8-channel pipette so that the samples occupy rows A, C, E, G, etc. Repeat with Gapdh directly under the SOD1 rows so that Gapdh samples occupy rows B, D, F, H, etc.; make sure to include standard curve (Figure 8).


        Figure 8. A few example columns of a qPCR plate map in a 384-well plate. Gapdh standard curve samples would be added to the final two columns in duplicate after all samples have been added.

      9. Spin at 400 x g for 3 min.
      10. Seal the plate with an optical plate sealer and run a standard-length TaqMan qPCR (Figure 9).


        Figure 9. Standard-length genotyping qPCR cycling conditions for qPCR machine setup

Data analysis

  1. Organize data
    1. Export the following data from the qPCR machine into Microsoft Excel: Raw Ct, Sample Name, Target Name.
    2. Find the average raw Ct of each set of duplicates.

  2. Find interpolated concentrations of unknowns using standard curve
    1. Open an XY spreadsheet in GraphPad Prism; choose “Enter X values” and “Enter and plot a single Y value for each point”.
    2. Label the X column “Concentration, ng/μl” and the first Y column “Gapdh Ct”.
    3. In the first 8 rows of the “Concentration”, put the concentration values of the standard curve (2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625).
    4. In the first 8 rows of the “Gapdh Ct” column, paste the raw average Ct values for your Gapdh standard curve.
    5. Click “Analyze” and choose “Nonlinear Regression” (If you need to choose again, choose semilog line–X is log, Y is linear”). Make sure “Interpolate unknowns from standard curve” is checked.
    6. Ensure that R-squared value is at least 0.99 in the Table of Results tab.
    7. Starting at Row 9, paste in raw average Gapdh Cts under “Gapdh Ct” column; a new Result tab will appear called “Interpolated X Values”.

  3. Normalize SOD1 Cts using interpolated concentrations
    1. Create a table in Excel with the following headings: Sample, Target Ct, Gapdh Concentration, Normalization Value, Normalization Coefficient, Log, Normalized SOD1 Ct.
    2. Use Figure 10A to put appropriate values into each column.

  4. Find relative copy number
    1. In a separate sheet, paste the Samples and their Normalized Target Ct values. Rank them from lowest to highest Ct using the Sort function, and assign them rank numbers starting at 1. If there are wild-types in your samples (Cts approaching 40), do not include them in this table. 
    2. Create a table in a separate Excel document with the following headings: Rank, Sample, Ct, Median, Ct-Median, Fold Change, Relative Copies.
    3. Use Figure 10B to put appropriate values into each column using your new ranked table.


      Figure 10. Excel table setup for Ct normalization and relative copy number calculation. A. Normalization of SOD1 Cts using interpolated Gapdh concentration. B. Relative copy number calculation.

  5. Make genotype calls
    1. As shown in the Background section, we found that 16 “relative copies” and above causes mice to have a normal lifespan. We make the “low copy” call for mice with 15 relative copies and fewer.
      Note: While we found that 15 copies were a safe threshold, you can be as strenuous as you want. For larger and higher-powered efficacy studies it may be worth it to change the threshold to 18 or 20 to make absolutely sure that no lower copy mice are put into the study.
    2. If you are analyzing stud tails, be more conservative because this affects the future of your colony. We use 18 as the cutoff for stud tails.
    3. It is worth keeping track of stud males for each cohort; if there is a large number of low copies in a cohort, they may all be the progeny of one low copy male.

  6. Representative Data
    Please see the attached Excel document “Representative Data Spreadsheet 08152018” for a representative set of data, including Excel formulas.

Notes

  1. Although no genotyping protocol is described in this paper, the mice were genotyped using this SOD1G93A genotyping protocol.
  2. There are many ways to arrive at high-quality genomic DNA; this method is written about our specific method, which has been optimized and customized for minimal cross-contamination,
  3. This protocol was written with this piece of equipment in mind; however, you can use another piece of equipment with comparable function.

Recipes

  1. Sample Prep solution


  2. Tissue Digestion solution


  3. Primer Dilution solution


  4. SOD1G93A Copy Number Genotyping qPCR Master Mix


  5. Primer/Probe solution

Acknowledgments

We acknowledge Matt Ferola and Carlos Maya for diligent and experienced mouse husbandry; we acknowledge Shawn Sullivan for his contribution to the acquisition of historical data. We thank Augie’s Quest for generous support of these studies. We thank people living with ALS for inspiring this research.

Competing interests

The authors declare no competing interests.

Ethics

All experiments were conducted in accordance with the protocols described by the National Institutes of Health Guide for the Care and Use of Animals and were approved by ALS TDI’s institutional animal care and use committee (IACUC). The IACUC ID is 2016-011 and the validity period is December 2016 through December 2019.

References

  1. Alexander, G. M., Erwin, K. L., Byers, N., Deitch, J. S., Augelli, B. J., Blankenhorn, E. P. and Heiman-Patterson, T. D. (2004). Effect of transgene copy number on survival in the G93A SOD1 transgenic mouse model of ALS. Brain Res Mol Brain Res 130(1-2): 7-15.
  2. Maier, M., Welt, T., Wirth, F., Montrasio, F., Preisig, D., McAfoose, J., Vieira, F. G., Kulic, L., Spani, C., Stehle, T., Perrin, S., Weber, M., Hock, C., Nitsch, R. M. and Grimm, J. (2018). A human-derived antibody targets misfolded SOD1 and ameliorates motor symptoms in mouse models of amyotrophic lateral sclerosis. Sci Transl Med 10(470). 
  3. Morrice, J. R., Gregory-Evans, C. Y. and Shaw, C. A. (2018). Animal models of amyotrophic lateral sclerosis: A comparison of model validity. Neural Regen Res 13(12): 2050-2054.
  4. Vieira, F. G., Hatzipetros, T., Thompson, K., Moreno, A. J., Kidd, J. D., Tassinari, V. R., Levine, B., Perrin, S. and Gill, A. (2017). CuATSM efficacy is independently replicated in a SOD1 mouse model of ALS while unmetallated ATSM therapy fails to reveal benefits. IBRO Rep 2: 47-53.
  5. Zwiegers, P., Lee, G. and Shaw, C. A. (2014). Reduction in hSOD1 copy number significantly impacts ALS phenotype presentation in G37R (line 29) mice: implications for the assessment of putative therapeutic agents. J Negat Results Biomed 13: 14.

简介

ALS临床前研究中最常用的小鼠模型表达人类SOD1(G93A)转基因的多个拷贝。在育种过程中,连续几代小鼠可以丢失转基因的拷贝。因为这些小鼠的寿命较短依赖于转基因拷贝数,所以必须确保在临床前研究中不包括低拷贝,因此寿命较长的小鼠。用于SOD1 G93A 小鼠基因分型的现有技术广泛地基于使用参考基因创建标准曲线并通过与标准曲线比较推断SOD1的相对量。这种技术用于Alexander et al。(2004),Vieira et al。(2017)和Maier et al。(2018) )。但是,没有详细描述(见注1)。本文提供了用于确定人SOD1转基因的相对拷贝数的详细方案。简而言之,该方案首先包括从小鼠耳组织中提取高质量的基因组DNA,基于基因组DNA浓度的标准曲线的创建,以及与Gapdh作为参考基因的标准曲线一起最多88个样品的qPCR分析。分析涉及使用标准曲线对每个未知样品进行标准化,然后确定相对于群组中值的样品的拷贝数。该方案已经过优化,可以生成高质量的基因组DNA和一致的结果,并且通过比较相对拷贝数和小鼠寿命,经验性地优化和验证了相对拷贝数截止值。
【背景】SOD1 G93A 小鼠模型(B6SJL-Tg(SOD1 * G93A)1Gur / J)是目前最常用的小鼠模型,用于肌萎缩侧索硬化症(ALS)的治疗前期临床试验,这是一种进行性神经退行性疾病大多数患者在确诊后生活2到5年(Morrice et al。,2018)。人SOD1转基因以多个拷贝存在,并且小鼠寿命与拷贝数反向相关。 Alexander et al。(2004)发现具有24个拷贝的SOD1(G93A)基因的小鼠活到大约129天,而具有34个拷贝的小鼠活到99天,具有20个拷贝的小鼠活到150天。天(Alexander et al。,2004)。此外,3.6%的高拷贝转基因雄性种鸡在4年的时间内具有低拷贝数的后代,这表明仅仅测试种马的拷贝数是不够的(Alexander et al。 ,2004)。虽然较高的拷贝确实与较短的寿命相关,但自发重组倾向于丢弃拷贝而不是增加拷贝数(Alexander et al。,2004; Zwiegers et al。,2014)。因此,我们将测试重点放在仅识别低拷贝小鼠上。

为了确保在临床前药物研究中的任何可测量的效果可归因于治疗而不是转基因拷贝的固有差异,绝对必要的是不包括低拷贝小鼠。虽然在专用方法或协议期刊中没有发表SOD1(G93A)基因分型方法,但有些论文在方法部分简要描述了基因分型。例如,Alexander et al。(2004)描述了一种基于qPCR的方法,该方法使用SOD1(G93A)的循环阈值(ΔCt)和参考基因的变化来量化拷贝数差异(Alexander et al。,2004)。在我们的手中,我们发现2016年之前发表的基因分型方法不够灵敏,不能排除所有低拷贝动物。图1显示了从2015年8月到2019年1月的非治疗组生存研究中所有小鼠的死亡年龄.X轴是每项研究的开始日期,每个开始日期的数据集是死亡包括在该研究的未治疗组中的小鼠的年龄。本文详述的最终基因分型方案于2016年5月9日制定,并且使用该方案进行基因分型的第一只小鼠被包括在该研究中,其开始日期为2016年6月7日。使用Box-在GraphPad Prism中分析该图。和 - Whiskers Tukey测试,突出显示任何值大于75 th 百分位数加上四分位数范围(IQR)的1.5倍或小于25 th 百分位减去的值IQR的1.5倍。这些异常值显示为大蓝点。由于基因分型方案已经实施,异常值的数量及其与中位数的距离趋于减少。


图1. 2015年8月至2019年1月小鼠死亡年龄的GraphPad Prism分析。这些小鼠处于未经处理的存活研究组。 x轴上显示的日期表示每个研究的开始日期,与每个日期相关的数据集表示每个研究的未处理组中小鼠的死亡年龄。 Tukey对盒须图的测试已经应用,蓝点显示哪些数据点超出了75 th 百分位数加上四分位数范围(IQR)的1.5倍或25 th 百分位数减去IQR的1.5倍。

自2016年以来,该基因分型方案已用于多项已发表的研究,包括Vieira et al。(2017),并提供了一种基于qPCR的方法,以确定最多88只小鼠耳的队列中的相对拷贝数。组织样本无需确定绝对基因拷贝。该方案依赖于基于浓度的Gapdh标准曲线来标准化样品。归一化后,根据每个样本和群组中位数之间的对数差异分配相对拷贝数。除了开发筛选低拷贝小鼠的可靠方法外,我们优化了组织收集,以避免交叉污染和优化基因组DNA提取,以获得稳定的产量和高质量(见注2)。此外,我们还进行了潜在参考基因引物和探针组的稀释qPCR,以确定哪个与定制SOD1靶引物/探针组最佳相关,并确定我们标准曲线的浓度范围(图2)。如绿色三角形所示,来自Thermo Fisher的拷贝数Gapdh引物/探针组具有与SOD1引物/探针组最相当的斜率和Ct值,并且两者在2.5和0.07ng /μl基因组DNA之间表现最佳。为了确定最安全的相对拷贝数截止值,我们进行了一项将拷贝数与寿命相关联的生存研究。具有16个或更多拷贝的小鼠显示该小鼠模型的典型寿命,而具有15个或更少拷贝的小鼠是长寿命的(图3)。使用这些信息,我们确定最安全的截止值为15份或更少。


图2.使用来自小鼠耳组织的SOD1 G93A 基因组DNA的连续两倍稀释液进行的qPCR实验,使用我们的定制SOD1引物/探针组以及几种非定型SOD1引物/探针组进行探测。来自Thermo Fisher的架子引物/探针分析。我们的定制SOD1引物/探针组与现成的SOD1分析相比较,Gapdh参考分析在Ct和斜率上与SOD1靶分析最接近线性区域。该图在0.07和2.5 ng /μl基因组DNA之间最为线性,这为我们的实验标准曲线提供了最佳范围。


图3.该图显示了SOD1 G93A 小鼠与基因分型方案指定的相对拷贝数相比的存活率。在可接受的死亡年龄范围内的小鼠是那些在16-23 +拷贝数组中。因此,我们选择15作为相对拷贝数的截止值。

关键字:ALS小鼠模型, 小鼠基因型, SOD1, 转基因拷贝数目, 临床前研究, 高通量基因分型, 相对拷贝数

材料和试剂

注意:除非另有说明,否则所有存储都处于室温下。

  1. QIAcube HT Plasticware(QIAGEN,产品目录号:950067)
  2. 粘合剂PCR板密封(Thermo Fisher,Thermo Scientific,目录号:AB0558)
  3. 无菌酒精预制垫(Fisher Scientific,Fisherbrand,目录号:22-363-750)
  4. 15 ml离心管(Corning,目录号:430766)
  5. 96孔紫外透明微孔板(康宁,目录号:3635)
  6. 384孔qPCR板(Thermo Fisher,Applied BioSystems,目录号:4309849) 
  7. 定制SOD1 MGB探针和引物(Thermo Fisher,Custom Genomics,-20°C)
    1. 探针:SOD1HumanV2-55T(VIC-ACTCTCTCCAACTTTG),具有VIC荧光报告化学,6000 pmole(湿,脱盐) 
    2. 正向引物:SOD1HumanV2-27F(GTAAATCAGCTGTTTTCTTTGTTCAGA),80,000 pmole(干燥,脱盐)
    3. 反向引物:SOD1HumanV2-104R(TTCACTGGTCCATTACTTTCCTTTAA),80,000 pmole(干燥,脱盐)
  8. QIAamp 96 QIAcube HT套件(QIAGEN,产品目录号:51331)
  9. PBS,磷酸盐缓冲盐水,10x溶液(Fisher BioReagents,Fisher Scientific,目录号:BP3994)
  10. 酒精,试剂(变性酒精),70%(v / v)水溶液(Ricca Chemical Company,Fisher Scientific,目录号:25467032,室温易燃品柜)
  11. Neosporin First Aid抗生素软膏(Hanna's Pharm Supply Co,目录号:302-571-8761)
  12. 棉花涂抹器(Fisher Scientific,Puritan,目录号:22-029-571)
  13. 50毫升试剂储存器(Corning,Costar,目录号:4870)
  14. DL-二硫苏糖醇溶液BioUltra用于分子生物学,1 M在H 2 O(Millipore Sigma,Sigma-Aldrich,目录号:43816),4°C
  15. 乙醇,绝对(200 Proof),分子生物学级(Fisher Scientific,目录号:BP2818100),室温易燃品柜
  16. 无核酸酶水(Thermo Fisher,Invitrogen,目录号:AM9932)
  17. TaqMan Genotyping Master Mix(Thermo Fisher,Applied Biosystems,目录号:4371355),4°C
  18. TaqMan Gapdh拷贝数测定(Thermo Fisher Assay ID Mm00186822cn),-20°C
  19. 样品制备溶液(参见食谱)
  20. 组织消化液(见食谱)
  21. 引物稀释溶液(见食谱)
  22. SOD1 G93A 拷贝编号基因分型qPCR Master Mix(见食谱)
  23. 引物/探针解决方案(见食谱)

设备

  1. 用于组织收获乙醇洗涤的两个灭菌容器
  2. 用于收集耳组织的手术质量工具(镊子和剪刀,或耳塞)
  3. 振荡器能够在200转/分钟和56℃温育18小时(Eppendorf,New Brunswick,目录号:M1352-0010)(见注3)
  4. Qiagen QIAcube HT(QIAGEN,目录号:9001793)(见注3)
  5. SpectraMax M5多功能微孔板读板机(Molecular Devices,目录号:M5)(见注3)
  6. NanoDrop TM 8000分光光度计(Thermo Fisher,Thermo Scientific,目录号:ND-8000-GL)(见注3)
  7. Allegra X-22R台式离心机(Beckman Coulter,目录号392188)(见注3)
  8. QuantStudio TM 7 Flex实时PCR系统,384孔,桌面(Thermo Fisher,目录号:4485701)(见注3)

软件

  1. Excel 2013(微软)
  2. 棱镜6(GraphPad, www.graphpad.com )

程序

  1. 组织收集
    注意:虽然该协议规定从耳朵样本中提取和分析基因组DNA,但也可以使用20 mg小鼠尾尖样本。
    1. 制备
      1. 创建一个样板图,其中样本从上到下按列组织(即。,样本1在点A1中,样本2在点B1中,等。)。请记住,在每个实验中,需要为标准曲线保留8个样本点,因此可以在单个qPCR中分析的大多数小鼠是88(图4A)。
      2. 创建适当过量(至少10)的样品制备溶液(配方1)。 
      3. 使用Qiagen QIAcube Plasticware试剂盒中的S-Block,将160μl样品制备溶液加载到实验所需的每个色谱柱的每个孔中(图4B)。
        注意:QIAcube HT以列格式工作,并且需要填充所使用的列的每个井(无论它们是否包含样本),以使自动协议正常工作。
      4. 用粘性板密封件密封S型块的顶部。


        图4.样品采集板示例。 A.样品采集板图,用于88样品实验,标准曲线的占位符孔。 B. Qiagen S-Block。

    2. 样品采集
      1. 用足够的70%试剂酒精填充两个干净的容器来清洗工具。
      2. 使用70%试剂酒精预清洁工具。
      3. 使用剪刀或耳孔从每只20毫克或更少的小鼠收集耳朵样品(图5);使用镊子,根据平板图将样品放入适当的孔中。
      4. 使用带有棉签的涂抹器在小鼠耳朵上涂抹抗生素软膏。
        注意:抗生素拭子通常不需要在使用之间进行清洁,但如果鼠标出血,我们会更换脏污的拭子。
      5. 在第一次酒精洗涤时漱口镊子和剪刀,用酒精预备垫擦拭去除任何固体材料如毛发,然后在第二次酒精洗涤中漱口。将工具留在第二次洗涤中,直到准备再次使用;不要混淆洗涤,因为第一次洗涤应起到“脏洗”的作用,第二次洗涤应起到“清洗”的作用。
      6. 对剩余的老鼠重复,经常更换酒精预备垫和清洁手套。
      7. 完成后以及任何长时间休息时,用粘合剂覆盖S块。


        图5.耳组织样本约为20毫克
    3. 样品消化
      1. 在试剂储库中,制备适当过量(至少10)的组织消化液(配方2),充分混合,无菌加载40μl到S-块的样品孔中;将配方加载到任何“空白”孔中(使用过的柱中没有样品的孔)。
      2. 确保每个样品都浸没在水中。
      3. 密封S型块,带有热稳定的粘性板盖;使用实验室胶带增加额外的安全性,以便在过夜消化过程中粘性覆盖物不会脱落(如果发生这种情况,样品会蒸发)。
      4. 固定在温度为56°C的热振荡器中;在200转/分钟下振荡过夜或最多18小时。

  2. 基因组DNA提取
    1. QIAcube HT设置
      1. 隔夜样品消化后,打开QIAcube HT的QIAamp 96协议。
      2. 按照QIAamp 96 QIAcube HT协议中的设置向导输入样品编号并进行以下自定义。
        1. 使洗脱体积为150μl(图6A)。
          注意:在我们的手中,150μl洗脱产生的样品产量在10-50 ng /μl之间,我们发现这是样品稀释的最佳范围。您可能需要更改洗脱体积以优化产量范围。 
        2. 删除Top Elute(将音量更改为0)(图6B)。
          注意:通过实验,我们发现使用Top Elute是混乱的,不会提高样品产量或质量。
      3. 根据软件程序的规格设置卡座。
      4. 在运行协议之前,用电离枪或风扇将甲板去离子。


        图6. QIAcube设置。 A.将洗脱缓冲液设置为150μl。 B.将Top Elute体积设置为0μl。

    2. 储存:协议完成后,用胶带和盖子盖住洗脱板,贴上标签,并在4°C下储存,直到准备使用。
      注意:该套件指示用户使用橡胶管帽,但我们发现这种情况很麻烦,并且液体进入另一口井的可能性增加。

  3. 基因组DNA稀释
    1. 使用平板读数器或MultiDrop读取DNA的260 nm,280 nm和320 nm读数(如果使用平板读数器,在UV平板中将gDNA 1:10稀释在水中)。
    2. 量化浓度和260/280比例;确保260/280值约为1.8。
      注意:另一个表示样品纯度的比率为260/230。理想的260/230值至少为1.0,但我们没有发现260/230值对qPCR结果有显着影响,因此在本协议中没有进一步解决。
    3. 找到浓度最高的样本;为整个板选择一个稀释因子,确保最高浓度样品不超过1 ng /μl,最低浓度样品不低于0.02 ng /μl。
    4. 用新板中的PCR-清洁水稀释所有样品并充分混合。

  4. 标准曲线设置
    1. 将由几种不同SOD1 G93A 小鼠制成的几个样品的高质量基因组DNA组合并混合至总共约100μl的混合样品。
    2. 找到浓度(理想情况下,使用NanoDrop获得最准确的结果);做一些重复以确保绝对。
    3. 用PCR-清水将样品稀释至80 ngl体积中的2 ng /μl;将剩余的混合gDNA原液保存在-20°C,以备将来的标准曲线使用。
    4. 从70μl的2 ng /μlgDNA开始,在PCR板的一列中的PCR-清水中进行8次连续两倍稀释;这样就形成了一条标准曲线,其中最浓缩的样品为2 ng /μl,最小浓度为0.016 ng /μl(图7)。
      注意:如果您经常使用它们,您可以一次制作多条标准曲线,并将它们存储在4°C。
    5. 用粘性板密封剂覆盖并在4°C下储存。每次使用标准曲线时都要使用无菌技术。


      图7. 96孔混合器板中的标准曲线设置

  5. qPCR设置
    1. 引物设置
      1. 根据“Primer Dilution solution”(配方3)稀释干燥的底漆;在板凳上离开10分钟,旋转,旋转下来。
      2. 将其等分成100μl等分试样,标记并在-20℃下储存。
      3. 将湿探针等分成20μl等分试样,标记并在-20°C下储存。
    2. qPCR Master Mix
      1. 根据“SOD1 G93A 拷贝数基因分型qPCR主混合物溶液”(配方4),使用适当的过量进行混合(确保在样品数量计算中包括标准曲线,并为两个基因做足够的, SOD1和Gapdh)。
      2. 使用适当的过量,根据“Primer / Probe solution”(配方5)创建SOD1和Gapdh混合物。请记住,在计算过量时,您可以使用最多用于SOD1和Gapdh混合物的主混合物的过量样品数量的一半。轻轻混合;储存在4°C,用箔覆盖,直到准备使用(不超过1-2小时)。
      3. 分别标记2个96孔PCR板“SOD1”和“Gapdh”。
      4. 将4μl稀释的未知gDNA等分到每个平板中。
      5. 在Gapdh板上的最后一列样品后加入4μl标准曲线。
      6. 将20μlSOD1主混合物等分到SOD1板的每个样品孔中;移液器混合得很好,但不会引入气泡。用Gapdh板和主混合物重复。
      7. 旋转板在180 x g 下旋转3分钟。
      8. 从SOD1板开始,用38通孔qPCR板用8通道移液管移取9.8μl复制品,使样品占据A,C,E,G,等行。直接用Gapdh重复在SOD1行下,使Gapdh样本占据行B,D,F,H,等。;确保包括标准曲线(图8)。


        图8. 384孔板中qPCR板图的几个示例列。 Gapdh标准曲线样品将在添加所有样品后一式两份加入到最后两列中。

      9. 在400 x g 下旋转3分钟。
      10. 用光学平板密封剂密封平板并运行标准长度的TaqMan qPCR(图9)。


        图9. qPCR机器设置的标准长度基因分型qPCR循环条件

数据分析

  1. 组织数据
    1. 将以下数据从qPCR机器导出到Microsoft Excel:Raw Ct,Sample Name,Target Name。
    2. 找出每组重复的平均原始Ct。

  2. 使用标准曲线查找插值浓度的未知数
    1. 在GraphPad Prism中打开XY电子表格;选择“输入X值”和“为每个点输入并绘制单个Y值”。
    2. 标记X列“浓度,ng /μl”和第一个Y列“Gapdh Ct”。
    3. 在“浓度”的前8行中,放置标准曲线的浓度值(2,1,0.5,0.25,0.125,0.0625,0.03125,0.015625)。
    4. 在“Gapdh Ct”列的前8行中,粘贴Gapdh标准曲线的原始平均Ct值。
    5. 单击“分析”并选择“非线性回归”(如果需要再次选择,请选择半对数线-X为对数,Y为线性“)。确保选中“从标准曲线插入未知数”。
    6. 确保“结果表”选项卡中的R平方值至少为0.99。
    7. 从第9行开始,在“Gapdh Ct”栏下粘贴原始平均Gapdh Cts;将出现一个名为“Interpolated X Values”的新结果选项卡。

  3. 使用插值浓度标准化SOD1 Cts
    1. 使用以下标题在Excel中创建一个表:Sample,Target Ct,Gapdh Concentration,Normalization Value,Normalization Coefficient,Log,Normalized SOD1 Ct。
    2. 使用图10A将适当的值放入每列中。

  4. 查找相对副本号
    1. 在单独的工作表中,粘贴“样本”及其“标准化目标Ct”值。使用Sort函数将它们从最低到最高Ct排名,并从1开始为它们分配等级数。如果样本中有野生型(Cts接近40),请不要在此表中包含它们。 
    2. 使用以下标题在单独的Excel文档中创建表:Rank,Sample,Ct,Median,Ct-Median,Fold Change,Relative Copies。
    3. 使用图10B,使用新排名表将适当的值放入每列。


      图10.用于Ct标准化和相对拷贝数计算的Excel表格设置。 A.使用插值Gapdh浓度对SOD1 Cts进行标准化。 B.相对拷贝数计算。

  5. 进行基因型调用
    1. 如背景部分所示,我们发现16“相对拷贝”及以上使小鼠具有正常寿命。我们对15个相对副本和更少的小鼠进行“低拷贝”调用。
      注意:虽然我们发现15个副本是一个安全的阈值,但您可以像您想要的那样费劲。对于更大和更高功效的研究,将阈值改为18或20可能是值得的,以确保没有较低的拷贝小鼠被纳入研究。
    2. 如果您正在分析螺柱尾部,请更加保守,因为这会影响您的殖民地的未来。我们使用18作为螺柱尾部的截止。
    3. 值得跟踪每个队列的男性角色;如果一个队列中有大量的低拷贝,它们可能都是一个低拷贝雄性的后代。

  6. 代表性数据
    请参阅随附的Excel文档“代表性数据电子表格08152018 “代表一组有代表性的数据,包括Excel公式。

笔记

  1. 尽管本文未描述基因分型方案,但使用该SOD1 G93A 基因分型方案对小鼠进行基因分型。
  2. 有很多方法可以获得高质量的基因组DNA;这种方法是关于我们的具体方法编写的,该方法经过优化和定制,可最大限度地减少交叉污染,
  3. 这个协议是用这件设备编写的;但是,你可以使用功能相当的另一件设备。

食谱

  1. 样品制备解决方案


  2. 组织消化液


  3. 引物稀释溶液


  4. SOD1 G93A 拷贝编号基因分型qPCR Master Mix


  5. 引物/探针解决方案

致谢

我们承认Matt Ferola和Carlos Maya的勤奋和经验丰富的鼠标饲养;我们感谢Shawn Sullivan对获取历史数据的贡献。我们感谢Augie's Quest对这些研究的慷慨支持。我们感谢与ALS一起生活的人们对这项研究的启发。

利益争夺

作者宣称没有竞争利益。

伦理

所有实验均按照美国国立卫生研究院动物护理和使用指南所述的方案进行,并由ALS TDI的机构动物护理和使用委员会(IACUC)批准。 IACUC ID为2016-011,有效期为2016年12月至2019年12月。

参考

  1. Alexander,G.M.,Erwin,K.L.,Byers,N.,Deitch,J.S.,Augelli,B.J.,Blankenhorn,E.P。和Heiman-Patterson,T.D。(2004)。 转基因拷贝数对ALS的G93A SOD1转基因小鼠模型中存活率的影响。 Brain Res Mol Brain Res 130(1-2):7-15。
  2. Maier,M.,Welt,T.,Wirth,F.,Montrasio,F.,Preisig,D.,McAfoose,J.,Vieira,FG,Kulic,L.,Spani,C.,Stehle,T.,Perrin ,S.,Weber,M.,Hock,C.,Nitsch,RM和Grimm,J。(2018)。 人源抗体靶向错误折叠的SOD1并改善肌萎缩侧索硬化小鼠模型的运动症状。< / a> Sci Transl Med 10(470)。&nbsp;
  3. Morrice,J.R.,Gregory-Evans,C.Y。和Shaw,C.A。(2018)。 肌萎缩侧索硬化的动物模型:模型有效性的比较。 Neural Regen Res 13(12):2050-2054。
  4. Vieira,F.G.,Hatzipetros,T.,Thompson,K.,Moreno,A.J.,Kidd,J.D.,Tassinari,V.R.,Levine,B.,Perrin,S。和Gill,A。(2017)。 CuATSM疗效在ALS的SOD1小鼠模型中独立复制,而未经金属化的ATSM疗法未能显示益处。 IBRO Rep 2:47-53。
  5. Zwiegers,P.,Lee,G。和Shaw,C.A。(2014)。 hSOD1拷贝数的减少显着影响G37R(第29行)小鼠的ALS表型呈现:对评估推定的治疗药物。 J Negat Results Biomed 13:14
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tassinari, V. R. and Vieira, F. G. (2019). A High-throughput qPCR-based Method to Genotype the SOD1G93A Mouse Model for Relative Copy Number. Bio-protocol 9(12): e3276. DOI: 10.21769/BioProtoc.3276.
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