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Minichromosomes are small, autonomously functioning chromosomes that exist separately from the normal chromosomal set. Creation of minichromosomes in plants relies on telomere truncation to remove the chromosome arms and the native telomere sequence and replace them with a transgene together with a new telomere sequence to generate a modifiable small chromosome. Telomere truncation has been accomplished utilizing both Agrobacterium tumefaciens, in which a telomere repeat sequence is cloned into the transformation vector near the right border, and particle bombardment, in which the genes of interest and telomere sequence are co-introduced into the plant. In this protocol we will describe the methods for introducing telomere sequences to both Agrobacterium and gold particles, as well as the methods required to verify that these sequences are intact.

[Introduction] Engineered minichromosomes are autonomously functioning chromosomes that contain all of the necessary components required for maintenance through the cell cycle. The production of engineered minichromosomes has several potential applications for the next generation of genetic engineering (Gaeta et al., 2012). The construction of such chromosomes by assembling a centromere, origin of replication, and selectable marker all capped by telomere sequences, as originally performed in yeast, is not feasible in plants because of the epigenetic nature of centromere sequences (Birchler and Han, 2009; Birchler et al., 2011; Liu et al., 2015). In other words, functional centromeres in plants are determined by chromatin features independent of the underlying DNA and therefore the cloning and re-introduction of centromere sequences will not produce a minichromosome. In contrast to the centromere, the telomere is reliant on sequence, with most plant telomeres containing the same TTTAGGG repeat (Adams et al., 2001). As a result, introduction of telomere sequences during transformation has the potential to confer telomere function.
Because of the epigenetic state of the centromere, engineered minichromosomes in plants need to be produced by cleaving away the chromosome arms from an endogenous centromere that never leaves a cell, a procedure known as the top-down method. This was accomplished with the finding that introduction of the chromosome end sequences, the telomere, would cleave chromosomes at the site of integration (Yu et al., 2006). By including genes of interest in addition to the telomere sequences, the foundation to build engineered minichromosomes to specification was established. This protocol describes the procedure to generate these initial truncated minichromosomes.
The process of creating minichromosomes utilizes standard plant transformation protocols. The only modification is the addition of telomere sequences to the transformation construction so that both a transgene and telomere sequence are introduced into a double stranded break during the transformation process. In some cases, the introduced telomere sequence is recognized by telomere elongation machinery and converted into a functioning telomere. As a result, the acentric fragment distal to the insertion point will be lost, and a minichromosome will be created.
Telomere truncation works well with both Agrobacterium tumefaciens and particle bombardment transformation techniques. Using Agrobacterium, successful minichromosome creation relies on inclusion of telomere sequences in the transformation construct. With particle bombardment, telomere sequences are simply added to the DNA mixture that is adhered to the gold beads before transformation is performed. While the length of telomere sequence required for telomere truncation is not known, a study in Arabidopsis thaliana successfully created truncated chromosomes with telomere repeats as short as 100 bp (Nelson et al., 2011). The study also found, however, that longer telomere sequences were more likely to induce truncation events. As a result, it is suggested that the largest amount of telomere that can be reasonably obtained be used during transformation.
While the concept of including telomere sequences in transformation is relatively simple, working with telomere sequences using current molecular cloning techniques is challenging. The repeated nature of the sequences and the high GC content are inhibitive to polymerase function. As a result, protocols reliant on polymerase function, such as PCR or Sanger sequencing, are not efficient. Additionally, oligonucleotide synthesis technologies are limited for producing telomere repeat sequences at the time of this writing. Traditional cloning utilizing restriction enzymes has been most successful in our work. Isolated telomere sequences, when subjected to agarose gels, do not migrate at the expected sizes, but instead are found as discreet bands or smears throughout the gel, probably because they adopt various secondary structures. Additionally, purification of telomere sequences with gel or column purification is usually inefficient unless the DNA is present in large amounts, making traditional cloning difficult. Adding to these challenges is the observation that long telomere sequences are unstable in microbial cells, and have a tendency to be deleted and shortened over time. As a result, Stbl cells (Invitrogen), which possess the recA1 genotype and are specifically designed to prevent repeated sequences from recombination and thus rearrangement, must be used to maintain the repeat, and multiple clones should be isolated and screened to ensure the full size is present. Additionally, when a clone has been isolated, which contains the desired telomere insert, it is often useful to make a large plasmid extraction that is stored in addition to bacterial stocks.
In order to generate engineered minichromosomes, the protocols presented below were developed. For cloning purposes, the telomere sequence is excised from a gel and ligated to the target plasmid within the agarose mixture. The source of the telomere sequence is plasmid pWY82 (Yu et al., 2006), which contains 2.6 kb of the telomere repeat (TTTAGGG). For particle bombardment, primers are used with a modified PCR program to amplify the telomere repeats, which are gel purified and added to gold particles together with the construct of interest. Whether truncation will be performed with Agrobacterium or particle bombardment, the standard transformation protocol for the species of interest can be followed. Fluorescence in-situ hybridization is then performed to determine if a minichromosome has successfully been produced (Yu et al., 2007).

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Telomere-mediated Chromosomal Truncation via Agrobacterium tumefaciens or Particle Bombardment to Produce Engineered Minichromosomes in Plants
通过农杆菌或粒子轰击法截断端粒介导染色体在植物中产生工程微型染色体

植物科学 > 植物分子生物学 > DNA > DNA 结构
作者: Nathaniel D. Graham*
Nathaniel D. GrahamAffiliation: University of Missouri Columbia, Tucker Hall, Columbia, USA
Bio-protocol author page: a2555
Nathan C. Swyers*
Nathan C. SwyersAffiliation: University of Missouri Columbia, Tucker Hall, Columbia, USA
Bio-protocol author page: a2556
Robert T. Gaeta
Robert T. GaetaAffiliation: University of Missouri Columbia, Tucker Hall, Columbia, USA
Bio-protocol author page: a2557
Changzeng Zhao
Changzeng ZhaoAffiliation: University of Missouri Columbia, Tucker Hall, Columbia, USA
Bio-protocol author page: a2558
Jon P. Cody
Jon P. CodyAffiliation: University of Missouri Columbia, Tucker Hall, Columbia, USA
Bio-protocol author page: a2559
 and James A. Birchler
James A. BirchlerAffiliation: University of Missouri Columbia, Tucker Hall, Columbia, USA
For correspondence: BirchlerJ@Missouri.edu
Bio-protocol author page: a2560
 (*共同第一作者)
Vol 5, Iss 18, 9/20/2015, 1765 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1595

[Abstract] Minichromosomes are small, autonomously functioning chromosomes that exist separately from the normal chromosomal set. Creation of minichromosomes in plants relies on telomere truncation to remove the chromosome arms and the native telomere sequence and replace them with a transgene together with a new telomere sequence to generate a modifiable small chromosome. Telomere truncation has been accomplished utilizing both Agrobacterium tumefaciens, in which a telomere repeat sequence is cloned into the transformation vector near the right border, and particle bombardment, in which the genes of interest and telomere sequence are co-introduced into the plant. In this protocol we will describe the methods for introducing telomere sequences to both Agrobacterium and gold particles, as well as the methods required to verify that these sequences are intact.

[Introduction] Engineered minichromosomes are autonomously functioning chromosomes that contain all of the necessary components required for maintenance through the cell cycle. The production of engineered minichromosomes has several potential applications for the next generation of genetic engineering (Gaeta et al., 2012). The construction of such chromosomes by assembling a centromere, origin of replication, and selectable marker all capped by telomere sequences, as originally performed in yeast, is not feasible in plants because of the epigenetic nature of centromere sequences (Birchler and Han, 2009; Birchler et al., 2011; Liu et al., 2015). In other words, functional centromeres in plants are determined by chromatin features independent of the underlying DNA and therefore the cloning and re-introduction of centromere sequences will not produce a minichromosome. In contrast to the centromere, the telomere is reliant on sequence, with most plant telomeres containing the same TTTAGGG repeat (Adams et al., 2001). As a result, introduction of telomere sequences during transformation has the potential to confer telomere function.
Because of the epigenetic state of the centromere, engineered minichromosomes in plants need to be produced by cleaving away the chromosome arms from an endogenous centromere that never leaves a cell, a procedure known as the top-down method. This was accomplished with the finding that introduction of the chromosome end sequences, the telomere, would cleave chromosomes at the site of integration (Yu et al., 2006). By including genes of interest in addition to the telomere sequences, the foundation to build engineered minichromosomes to specification was established. This protocol describes the procedure to generate these initial truncated minichromosomes.
The process of creating minichromosomes utilizes standard plant transformation protocols. The only modification is the addition of telomere sequences to the transformation construction so that both a transgene and telomere sequence are introduced into a double stranded break during the transformation process. In some cases, the introduced telomere sequence is recognized by telomere elongation machinery and converted into a functioning telomere. As a result, the acentric fragment distal to the insertion point will be lost, and a minichromosome will be created.
Telomere truncation works well with both Agrobacterium tumefaciens and particle bombardment transformation techniques. Using Agrobacterium, successful minichromosome creation relies on inclusion of telomere sequences in the transformation construct. With particle bombardment, telomere sequences are simply added to the DNA mixture that is adhered to the gold beads before transformation is performed. While the length of telomere sequence required for telomere truncation is not known, a study in Arabidopsis thaliana successfully created truncated chromosomes with telomere repeats as short as 100 bp (Nelson et al., 2011). The study also found, however, that longer telomere sequences were more likely to induce truncation events. As a result, it is suggested that the largest amount of telomere that can be reasonably obtained be used during transformation.
While the concept of including telomere sequences in transformation is relatively simple, working with telomere sequences using current molecular cloning techniques is challenging. The repeated nature of the sequences and the high GC content are inhibitive to polymerase function. As a result, protocols reliant on polymerase function, such as PCR or Sanger sequencing, are not efficient. Additionally, oligonucleotide synthesis technologies are limited for producing telomere repeat sequences at the time of this writing. Traditional cloning utilizing restriction enzymes has been most successful in our work. Isolated telomere sequences, when subjected to agarose gels, do not migrate at the expected sizes, but instead are found as discreet bands or smears throughout the gel, probably because they adopt various secondary structures. Additionally, purification of telomere sequences with gel or column purification is usually inefficient unless the DNA is present in large amounts, making traditional cloning difficult. Adding to these challenges is the observation that long telomere sequences are unstable in microbial cells, and have a tendency to be deleted and shortened over time. As a result, Stbl cells (Invitrogen), which possess the recA1 genotype and are specifically designed to prevent repeated sequences from recombination and thus rearrangement, must be used to maintain the repeat, and multiple clones should be isolated and screened to ensure the full size is present. Additionally, when a clone has been isolated, which contains the desired telomere insert, it is often useful to make a large plasmid extraction that is stored in addition to bacterial stocks.
In order to generate engineered minichromosomes, the protocols presented below were developed. For cloning purposes, the telomere sequence is excised from a gel and ligated to the target plasmid within the agarose mixture. The source of the telomere sequence is plasmid pWY82 (Yu et al., 2006), which contains 2.6 kb of the telomere repeat (TTTAGGG). For particle bombardment, primers are used with a modified PCR program to amplify the telomere repeats, which are gel purified and added to gold particles together with the construct of interest. Whether truncation will be performed with Agrobacterium or particle bombardment, the standard transformation protocol for the species of interest can be followed. Fluorescence in-situ hybridization is then performed to determine if a minichromosome has successfully been produced (Yu et al., 2007).
Keywords: Artificial chromosomes(人工染色体), Synthetic chromosomes(合成染色体), Telomere truncation(端粒截断), Genetic engineering(遗传工程), Gene stacking(基因叠加)

[Abstract]

Materials and Reagents

  1. Target binary plasmid with compatible restriction enzyme cut sites near right border or purified plasmid for co-bombardment
    Note: There are many binary vectors available, and any are acceptable for use provided the vector contains the necessary restriction enzyme cut sites to move the telomere fragment from pWY82. A map and sequence of pWY82 can be obtained by contacting the corresponding author. In addition, it is suggested to place the telomere sequences near the right border as in the original truncation plasmid (Yu et al., 2007). It is not known whether placing it inside the left border is effective.
  2. Plasmid pWY82 (Contact corresponding author)
  3. QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27104 ) 

  4. AmbionTM Nuclease-Free Water (not DEPC-Treated) (Fisher Scientific, catalog number: AM9937 ) 

  5. Restriction Enzymes (New England Biolabs)
    Note: Enzymes must be chosen based on compatibility between pWY82 and target vector.

  6. UltraPureTM Low Melting Point Agarose (Life Technologies, InvitrogenTM, catalog number: 16520-050 ) 

  7. Trizma® base (Sigma-Aldrich, catalog number: T1503 )

  8. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 ) 

  9. Acetic Acid (Sigma-Aldrich, catalog number: 27225 ) 

  10. Antarctic Phosphatase (New England BioLabs, catalog number: M0289S ) 

  11. DNA Gel Loading Dye (6x) (Thermo Fisher Scientific, catalog number: R0611 ) 

  12. GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific, catalog number: SM0311 ) 

  13. Ethidium Bromide (Sigma-Aldrich, catalog number: E7637 ) 

  14. T4 DNA Ligase (New England BioLabs, catalog number: M0202S ) 

  15. ElectroMAX Stbl4 Cells (、Thermo Fisher Scientific, catalog number: 11635-018 ) 

  16. S.O.C Media (Super Optimal Broth with Catabolic repressor) (Thermo Fisher Scientific, catalog number: 15544-034 ) 

  17. Agar (Sigma-Aldrich, catalog number: A1296 ) 

  18. Petri Dishes 

  19. LongAmp® Taq DNA Polymerase (New England BioLabs, catalog number: M0323S )

  20. Fisher BioReagents LB Broth, Miller (Granulated) (Fisher Scientific, catalog number: BP9723-2 ) 

  21. Spectinomycin dihydrochloride pentahydrate (Sigma-Aldrich, catalog number: S4014 )

  22. 2x YT medium (Sigma-Aldrich, catalog number: Y2377 ) 

  23. LB broth (see Recipes)
  24. LB plates (see Recipes)
  25. 2x YT broth (see Recipes)
  26. Spectinomycin (see Recipes)
  27. TAE (see Recipes)

Equipment

  1. 30 °C incubator 

  2. 30 °C shaker 

  3. 4 °C cold room 

  4. 250 ml baffled culture flasks 

  5. Nanodrop spectrophotometer (Thermo Fisher Scientific) 

  6. Vacuum concentrator 

  7. Gel electrophoresis system 

  8. 37 °C waterbath 

  9. 70 °C waterbath 

  10. Ultraviolet transilluminator 

  11. Electroporator 

  12. Thermalcycler
  13. Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, catalog number: A9281 )

Procedure

  1. In-Gel ligation
    1. Streak a LB plate containing 100 mg/ml spectinomycin with plasmid pWY82 and grow at 30 °C for 2 days. 

    2. Begin a 3 ml starter culture with a single colony of pWY82 in 2XYT media with 100 mg/ml spectinomycin and grow for 48 h at 30 °C and 250 rpm.
      Note: pWY82 is a slow growing plasmid. 

    3. Add 500 μl of starter culture to 125 ml 2XYT in a baffled culture flask. Shake at 250 rpm for 48 h at 30 °C until OD600 = ~2 when measured with a spectrophotometer.
    4. Extract plasmid DNA with the QIAprep Spin Miniprep kit, using 4 ml 
of culture per spin column. Elute with 50 μl of nuclease free water preheated 
to 50 °C. 

    5. Pool minipreps into a 1.7 ml tube and place into a vacuum concentrator until 
the concentration is about 1 μg/μl when measured with a Nanodrop spectrophotometer. 

    6. Confirm the integrity of pWY82, target plasmid, and cut sites by digesting 1 μg of pWY82 and target plasmid with each enzyme individually and in tandem following the manufacturer’s instructions.
      Note: Due to the instability of telomere repeats, the telomere fragment will show multiple bands when subjected to agarose gel electrophoresis with the full 2.6 kb repeat not always visible (Figure 1).


      Figure 1. 1% (w/v) agarose gel of three different colonies of pWY82 cut with EcoRI and HindIII showing the range of telomere sizes

    7. Digest 10 μg of pWY82 and 5 μg of target plasmid with 10 units of each restriction enzyme for at least one hour. 

      Note: Total restriction digest volume should be no more than 50 μl. 

    8. Pour a 1% (w/v) low melting point agarose gel prepared with 1x TAE in a 4 °C cold room. 

      Note: Use a comb large enough to fit each restriction digest fully in a single well.
    9. Treat target plasmid with 5 units of Antarctic phosphatase for 15 min at 
37 °C. 

    10. Add 15 μl of 6x loading dye to each reaction. 

    11. Fill the electrophoresis chamber with cold 1x TAE and carefully insert the 
gel.
      
Caution: Low melting point gels are extremely fragile. 

    12. Load each restriction digest completely into the gel. 

    13. Load 6 μl of GeneRuler 1 kb DNA Ladder next to each restriction digest lane.
    14. Run the gel in a 4 °C cold room at 100 V until the lower band of loading dye 
has reached the bottom of the gel. 

    15. Carefully transfer the gel to a dish containing 1x TAE and 0.5 μg /ml ethidium bromide and stain for 30 min. 

    16. Visualize DNA with UV light and estimate concentrations by comparison to the reference DNA ladder.
      Note: Concentration is estimated by comparing sample UV intensity with ladder UV intensity. The amount of DNA present in the ladder is available in the literature. 

    17. Carefully excise the uppermost visible telomere band and the target plasmid backbone with a scalpel and place into separate 1.7 ml tubes (Figure 2). 



      Figure 2. Example of gel to be used for ligation. pNG4 was the target plasmid for this ligation. The boxed sections were excised.

    18. Add 1 ml of nuclease free water to gel slices and allow to dialyze at 4 °C overnight to remove salts from agarose. 

    19. Remove water from tubes and place in a 70 °C water bath until completely melted (about 5 min).
      Note: Flick tubes each minute to ensure agarose melts completely. 

    20. Once melted, move tubes to 37 °C water bath. 

    21. Prepare ligase mixture in a separate 1.7 ml tube while agarose cools (Table 1).

      Table 1. Ligation reaction components 

      Component
      Amount
      Ligase buffer
      5 μl
      T4 DNA ligase
      1 μl (400 units)
      Target plasmid backbone
      100 ng as estimated from gel
      Telomere
      To 50 μl
    22. After cooling about 5 min, add target plasmid and telomere to ligation 
mixture. 

      Note: Work quickly so agarose does not solidify in pipette tip.
    23. Mix by gentle pipetting and allow to return to room temperature. 

      Note: The reaction should solidify as it cools. 

    24. Incubate at room temperature overnight. 

    25. Dialyze reaction by adding 1 ml of water to tube and incubate at room 
temperature for 15 min.

      Note: Flick tube until agarose is freely floating in water. 

    26. Begin to thaw Stbl4 cells on ice.
      Note: 40 μl of cells are needed per reaction.
    27. Completely remove water from dialysis and add 50 μl of fresh nuclease free 
water. Place tube in a 70 °C water bath for 10 min, or until completely melted.
      
Note: Gently flick tube every minute to ensure gel is completely 
melted. 

    28. Add 2 μl of ligation reaction to 40 μl of Stbl4 cells and electroporate following 
manufacturers recommended settings for the electroporator. Resuspend electroporated cells in 500 ml of S.O.C media and shake in 15 ml culture tubes for 1.5 h at 250 rpm at 30 °C. 

    29. Plate 100 μl of the transformation preparation onto pre-warmed LB plates containing the appropriate antibiotic and incubate at 30 °C until colonies appear. 

      Note: Due to the lower temperature, colonies may take 2 days to 
appear on plates. 

    30. Screen for positive colonies with either colony PCR or colony hybridization. 

      Note: Screening for positive colonies: 

      Due to the repetitive nature of the telomere, it is not advisable to screen colonies by amplifying across the telomere insertion site, as most PCR reactions will fail. Instead, colony PCR can be carried out using a forward primer that anneals 5’ to the insertion site in the target plasmid, and a reverse primer that anneals to a site 5’ to the telomere repeat in pWY82 that is also inserted into the target plasmid as shown in Figure 3 below. It is important to ensure good primer function in each plasmid before attempting colony PCR. The primer pWY82R with the sequence 5’ GTGGGACCCGAGGGAATC 3’ has been routinely used as the reverse primer in colony PCR.


      Figure 3. Primers for colony PCR should be chosen that border the insertion point, and do not amplify the telomere repeat

      Alternatively, entire plates of colonies can be screened for the presence of telomere by colony hybridization (Sambrook et al., 1989). This method allows for rapid screening of hundreds of colonies at once, and is extremely sensitive. The oligonucleotide probe used for the hybridization consists of 10 units of the telomere repeat, either in the 5’ or 3’ direction. Finally, it is suggested to sequence into the telomere repeat to ensure that the insert is in the correct orientation.

  2. Detecting telomere insert size
    Once positive colonies have been obtained, it is important to screen colonies for telomere size. For most purposes, the colony with the largest telomere insert possible is desired for transformation. Telomere size estimation is best carried out through Southern transfer and hybridization (Southern, 1975). The target plasmid, as well as plasmid pWY82, is run alongside the tested colonies to serve as controls.
    1. Inoculate positive colonies, plus one culture of pWY82 and one culture of original target plasmid, into 3 ml of 2XYT media containing the appropriate antibiotics. Shake culture at 250 rpm at 30 °C for 8 h. 

    2. Inoculate a fresh 5 ml 2XYT culture with 100 μl of starter culture of each colony containing the appropriate antibiotics and shake at 250 rpm for 16-18 h at 30 °C. 

      Note: It is sometimes useful to inoculate and grow larger (25 ml) cultures of colonies that have been chosen. An aliquot can be used for screening, and when a clone has been identified with the desired telomere fragment, the remaining culture can then be used for a larger scale plasmid preparation. 

    3. Using 4 ml of each culture, extract plasmid DNA with the QIAprep Spin Miniprep Kit. Elute plasmid extraction into 40 μl of warm (55 °C) nuclease free water. 

    4. Perform a restriction digest on 1 μg of each plasmid in a 20 μl total volume using enzymes that will cut as close to the telomere insert as possible on both ends so the telomere insert is excised. Additionally, cut pWY82 with enzymes that will also excise telomere insert for reference. 

    5. Pour a standard 1% (w/v) 1x TAE agarose gel using combs large enough to fit the entire restriction digest. 

    6. Once the restriction digest is complete, add 5 μl 6x loading dye to each reaction. 

    7. Load each digest into the agarose gel and run at 100 V until the lower band has reached the bottom of the gel. 

    8. Carefully transfer the gel to a dish containing 1x TAE and 0.5 μg/ml ethidium bromide and stain for 30 min. 

    9. Visualize DNA with UV light.

      Note: Due to the secondary structure of telomere, estimation of telomere size from a restriction digest will be unreliable. 

    10. Transfer the DNA to a nitrocellulose membrane by Southern transfer (Green and Sambrook, 2012). 

    11. Hybridize the membrane with a radiolabeled oligonucleotide and compare to the size standard to estimate telomere size (Green and Sambrook, 2012). 

      Note: An oligonucleotide of sequence (TTTAGGG)10 is extremely sensitive for detecting the presence of telomere. 


  3. Telomere PCR protocol to generate telomere fragments
    Telomere fragments of varying sizes can be generated using PCR for use in the co-bombardment method of minichromosome production. With the primers noted below, and running the reaction as shown, will generate a variety of telomere conglomerates of differing sizes can be produced by cross annealing of the primers. These telomere lengths can be visualized by gel electrophoresis. Telomere DNA of a particular length can be obtained by cutting out the size of telomere desired from the agarose gel. These steps below will outline how to generate telomere fragments via PCR and how to utilize the DNA obtained. This protocol is adapted from a similar protocol used to make fluorescent probes that label telomere (IJdo et al., 1991).
    1. Develop PCR primers
      1. Use the sequences below for primer development. These will allow for the production of large telomere fragments.
      2. Telomere primer sense: 5’ (TTTAGGG)10 3’.
      3. Telomere primer antisense: 5’ (CCCTAAA)10 3’
      4. Primers can be diluted to the micromolar concentrations listed on the table below which result in differing telomere fragment sizes.

      1.25 micromoles of Forward Telomere Primer
      1.25 micromoles of Reverse Telomere Primer
      Produces Larger Fragments (see Figure 4)
      25 micromoles of Forward Telomere Primer
      25 micromoles of Reverse Telomere Primer
      Produces Shorter Fragments (see Figure 4)

    2. PCR reaction assembly

      1. Use a proofreading Taq polymerase, such as LongAmp Taq DNA Polymerase from New England Biolabs, and use the above concentration of primers. At least four reactions are recommended in order to obtain enough DNA for use after DNA gel extraction. PCR reaction assembly is as follows:
        PCR Reaction Assembly using LongAmp Taq Polymerase from NEB

        Nuclease Free Water
        12 microliters
        24 microliters
        5x LongAmp Taq Buffer
        4 microliters
        8 microliters
        Sense Telomere Primer
        0.5 microliters
        1 microliter
        Antisense Telomere Primer
        0.5 microliters
        1 microliter
        10mM dNTPs
        2 microliters
        4 microliters
        LongAmp Taq Polymerase
        1 microliters
        2 microliters

        20 microliters per reaction
        40 microliters per reaction

        Thermocycler protocol
        98 °C 10 sec
        55 °C 20 sec
        72 °C 5 sec
        Repeat 5x
        98 °C 20 sec
        55 °C 20 sec
        72 °C 5 sec
        Repeat 5x
        98 °C 20 sec
        55 °C 20 sec
        72 °C 10 sec
        Repeat 5x
        98 °C 20 sec
        62 °C 20 sec
        72 °C 30 sec
        Repeat 5x
        72 °C 5 min

        4 °C ∞


      Note: It is recommended that either large volume PCR reactions are used or several PCR reactions are prepared to increase the amount of telomere DNA available for use.
    3. Use gel electrophoresis to determine the size of fragments generated (Figure 4).
      Notes:
      1. The smears of stained DNA on the gel image in Figure 4 are telomere fragments of varying size. Figure 4 is the result of 40 microliter PCR reactions. Cutting out the smear associated with the desired size indicated by the ladder provides telomere fragments of that length.
      2. The smears of telomere fragments in Figure 4 vary in size based on the Taq polymerase used as well as the primer concentration.


        Figure 4. Example of Telomere PCR product imaged after gel electrophoresis: L indicates the 1kb ladder used for this experiment. 1.25 micromoles and 25 micromoles indicate the concentration of PCR primer present in each sample.

      3. Follow the gel electrophoresis protocol (Sambrook and Russell, 2001).
    4. Gel extraction of telomere DNA

      1. Excise the piece of gel corresponding to the size of telomere desired, and extract the telomere DNA from this gel piece. For example, if a 2 kb telomere fragment is desired, cut out the gel smear closest in relation to the 2 kb mark on the ladder. Generally, larger fragments of telomere are desired if the goal is truncation of chromosomes because the larger the telomere fragment, the better chance that an insertion of the telomere fragment will be recognized by the telomere capping machinery as the new end for the chromosome. The minimum size of telomere fragment required for this recognition may vary from species to species (Teo et al., 2011).

      2. Use the Wizard SV Gel and PCR Clean-Up System for extracting DNA from agarose gels. Follow the manufacturer’s instructions for extracting DNA from the agarose gel piece.

        Notes:
        1. A significant amount of DNA will be lost when performing gel extraction. It is recommended to use a large volume PCR reaction or multiple reactions.

        2. It is recommended that elutions are performed using nuclease-free water, such as Nuclease-Free Water from Life Technologies, to allow for further use in cloning if desired.
    5. Using the extracted DNA
      1. Telomere DNA fragments obtained can then be used in a cobombardment with a transgene.
      2. Preparation of telomere DNA as described in (Kikkert et al., 2005) can be used in a co-bombardment.

  4. Discussion
    Conceptually, minichromosomes can be created by either a bottom-up or a top-down strategy. A bottom-up strategy would require the artificial creation of centromere, telomere, and origin of replication sequences to create a viable chromosome. Currently, origins of replication and centromeres cannot be made artificially in most eukaryotes. The top-down approach utilizes an existing chromosome to create a minichromosome by truncating it with a telomere-containing construct. Telomere-mediated truncation is an effective means of creating top-down minichromosomes. The essential factor for telomere-mediated truncation, the telomere sequences, can be difficult to manipulate because of their constitution as a large array of repeats. The methods for creating and utilizing telomere DNA described here are the most effective. After cloning telomere into a vector, or preparing telomere for cobombardment, proceed normally with transformation protocols. Tissue regeneration is the same as for standard transformation. Selection for truncation events can be performed using cytological techniques, such as fluorescent in situ hybridization (Yu et al., 2007). These methodologies should allow for the creation of telomere DNA of desired size, inclusion into a vector if so desired, introduced into the species of choice, and selection for truncated chromosomes.

Recipes

  1. LB broth
    For 500 ml of culture media, dissolve 12.5 g of LB media in 400 ml of water
    Bring final volume to 500 ml and autoclave for 20 min

  2. LB plates
    For 500 ml of media, dissolve 12.5 g of LB media and 6 g of agar in 400 ml of water
    Bring final volume to 500 ml and autoclave for 20 min
    Allow to cool to 50 °C and add appropriate antibiotics
    Gently swirl and add thin layer to petri dishes
    Allow to cool then invert and store at 4 °C
  3. 2x YT broth
    For 500 ml of culture media dissolve 15.5 g of 2x YT powder in 400 ml of water
    Bring final volume to 500 ml and autoclave for 20 min

  4. Spectinomycin (100 mg/ml)
    For 10 ml of stock, add 1 g of spectinomycin to 10 ml of autoclaved water in a 15 ml conical tube
    Invert until completely dissolved and filter sterilize with a 0.2 micron filter
    Aliquot 500 μl into 1.7 ml tubes and store at -20 °C
  5. TAE
    To make 1 L of 50x TAE stock add 242 g trizma base, 14.6 g ethylenediaminetetraacetic acid (EDTA), and 57.1 ml of acetic acid to 500 ml of water and dissolve
    Bring total volume to 1 L with water
    Dilute to 1x working stock with water

Acknowledgements

Research on this topic was supported by National Science Foundation grant IOS-1339198 from the Plant Genome Program.

References

  1. Adams, S. P., Hartman, T. P., Lim, K. Y., Chase, M. W., Bennett, M. D., Leitch, I. J. and Leitch, A. R. (2001). Loss and recovery of Arabidopsis-type telomere repeat sequences 5'-(TTTAGGG)(n)-3' in the evolution of a major radiation of flowering plants. Proc Biol Sci 268(1476): 1541-1546.
  2. Birchler, J. A., Gao, Z., Sharma, A., Presting, G. G. and Han, F. (2011). Epigenetic aspects of centromere function in plants. Curr Opin Plant Biol 14(2): 217-222.
  3. Birchler, J. A. and Han, F. (2009). Maize centromeres: structure, function, epigenetics. Annu Rev Genet 43: 287-303.
  4. Gaeta, R. T., Masonbrink, R. E., Krishnaswamy, L., Zhao, C. and Birchler, J. A. (2012). Synthetic chromosome platforms in plants. Annu Rev Plant Biol 63: 307-330.
  5. Green, M.R., and Sambrook, J. (2012). Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press.
  6. Ijdo, J. W., Wells, R. A., Baldini, A. and Reeders, S. T. (1991). Improved telomere detection using a telomere repeat probe (TTAGGG)n generated by PCR. Nucleic Acids Res 19(17): 4780.
  7. Kikkert, J. R., Vidal, J. R. and Reisch, B. I. (2005). Stable transformation of plant cells by particle bombardment/biolistics. Methods Mol Biol 286: 61-78.
  8. Nelson, A. D., Lamb, J. C., Kobrossly, P. S. and Shippen, D. E. (2011). Parameters affecting telomere-mediated chromosomal truncation in Arabidopsis. Plant Cell 23(6): 2263-2272.
  9. Sambrook, J., and Russell, D.W. (2001). Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory Press.
  10. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press.
  11. Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98(3): 503-517.
  12. Teo, C. H., Ma, L., Kapusi, E., Hensel, G., Kumlehn, J., Schubert, I., Houben, A. and Mette, M. F. (2011). Induction of telomere-mediated chromosomal truncation and stability of truncated chromosomes in Arabidopsis thaliana. Plant J 68(1): 28-39.
  13. Vega, J. M., Yu, W., Han, F., Kato, A., Peters, E. M., Zhang, Z. J. and Birchler, J. A. (2008). Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors. Plant Mol Biol 66(6): 587-598.
  14. Yu, W., Lamb, J. C., Han, F. and Birchler, J. A. (2006). Telomere-mediated chromosomal truncation in maize. Proc Natl Acad Sci U S A 103(46): 17331-17336.
  15. Yu, W., Han, F., Gao, Z., Vega, J. M. and Birchler, J. A. (2007). Construction and behavior of engineered minichromosomes in maize. Proc Natl Acad Sci U S A 104(21): 8924-8929.

材料和试剂

  1. 目标二元质粒与相邻的限制酶切割位点靠近右边界或纯化质粒共同轰击 注意:有许多可用的二元载体,任何可以使用的,只要载体含有必要的限制酶切割位点,以移动pWY82的端粒片段。 pWY82的地图和序列可以通过联系相应的作者获得。另外,建议将端粒序列置于右边界附近,如原始截短质粒(Yu等,2007)。将其放在左边框内是不是有效的。
  2. 质粒pWY82(联系对应作者)
  3. QIAprep Spin Miniprep Kit(QIAGEN,目录号:27104)
  4. Ambion TM 无核酸酶水(未经DEPC处理)(Fisher Scientific,目录号:AM9937)
  5. 限制酶(New England Biolabs)
    注意:酶必须基于pWY82和靶向量之间的兼容性来选择。
  6. UltraPure TM 低熔点琼脂糖(Life Technologies,Invitrogen TM,目录号:16520-050)
  7. Trizma ®(Sigma-Aldrich,目录号:T1503)
  8. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
  9. 乙酸(Sigma-Aldrich,目录号:27225)
  10. 南极磷酸酶(New England BioLabs,目录号:M0289S)
  11. DNA凝胶加载染料(6x)(Thermo Fisher Scientific,目录号:R0611)
  12. GeneRuler 1 kb DNA梯(Thermo Fisher Scientific,目录号:SM0311)
  13. 溴化乙锭(Sigma-Aldrich,目录号:E7637)
  14. T4 DNA连接酶(New England BioLabs,目录号:M0202S)
  15. ElectroMAX Stbl4细胞(Thermo Fisher Scientific,目录号:11635-018)
  16. S.O.C Media(具有Catabolic阻遏物的超级最佳肉汤)(Thermo Fisher Scientific,目录号:15544-034)
  17. 琼脂(Sigma-Aldrich,目录号:A1296)
  18. 培养皿
  19. DNA聚合酶(新英格兰BioLabs,目录号:M0323S)的Taq
  20. Fisher BioReagents LB Broth,Miller(Granulated)(Fisher Scientific,目录号:BP9723-2)
  21. 倍他霉素二盐酸盐五水合物(Sigma-Aldrich,目录号:S4014)
  22. 2x YT培养基(Sigma-Aldrich,目录号:Y2377)
  23. LB肉汤(见食谱)
  24. LB板(参见食谱)
  25. 2x YT肉汤(见食谱)
  26. 壮观霉素(见食谱)
  27. TAE(见食谱)

设备

  1. 30℃培养箱
  2. 30°C振动筛
  3. 4°C冷室
  4. 250ml带挡板的培养瓶
  5. Nanodrop分光光度计(Thermo Fisher Scientific)
  6. 真空浓缩机
  7. 凝胶电泳系统
  8. 37°C水浴
  9. 70°C水浴
  10. 紫外透射仪
  11. 电动机
  12. 热循环器
  13. 向导® SV凝胶和PCR清洁系统(Promega Corporation,目录号:A9281)

程序

  1. 凝胶内结扎
    1. 用含有质粒pWY82的含有100mg/ml壮观霉素的LB平板条纹,并在30℃下生长2天。
    2. 在2XYT中用单个菌落的pWY82开始3ml起始培养物 培养基中加入100毫克/毫升壮观霉素,在30℃和250℃下生长48小时 rpm。
      注意:pWY82是缓慢生长的质粒。
    3. 加入500μl 起始培养至125ml 2XYT的带挡板的培养瓶中。摇动250 在30℃下转速48小时,直到用a测量时,OD 600为〜2 分光光度计。
    4. 用QIAprep Spin提取质粒DNA Miniprep试剂盒,使用4毫升 的每个纺丝柱的培养。用50μl洗脱 的核酸酶自由水预热 至50°C。
    5. 泳池小型进入  将1.7ml管放入真空浓缩器中直到 的 当用Nanodrop测量时,浓度约为1μg/μl 分光光度计。
    6. 确认pWY82的完整性,目标 质粒和切割位点,通过消化1μgpWY82和靶质粒 每个酶单独和串联在制造商之后  说明。
      注意:由于端粒重复的不稳定性, 当进行琼脂糖时,端粒片段将显示多个条带  凝胶电泳与完整的2.6 kb重复不总是可见 (图1)。


      图1. 1%(w/v)琼脂糖凝胶三种不同 pWY82的菌落用Eco Eco RI切割,并且显示范围为 端粒大小

    7. 将10μgpWY82和5μg靶标质粒用10个单位的限制酶消化至少1小时。
      注意:总限制性消化量不应超过50μl。
    8. 倒入1℃(w/v)低温琼脂糖凝胶,用1×TAE在4℃的冷室中制备。
      注意:使用足够大的梳子将每个限制性消化装置完全装入单个孔中。
    9. 用5单位的南极磷酸酶处理目标质粒15分钟 37°C。
    10. 在每个反应中加入15μl6x负载染料。
    11. 在电泳室中加入1×TAE并小心地灌注 插入 凝胶。
      注意:低熔点凝胶非常脆弱。
    12. 将每个限制性消化内容物完全装入凝胶。
    13. 在每个限制性消化道旁边加载6μlGeneRuler 1 kb DNA梯子。
    14. 在4℃的冷室中以100V的速度运行凝胶,直到加载染料的下段 已经到达了凝胶的底部。
    15. 小心地将凝胶转移到含有1 TAE和0.5μg/ml溴化乙锭的培养皿中并染色30分钟。
    16. 用紫外光可视化DNA,并通过与参考DNA梯相比估计浓度。
      注意:通过比较样品UV强度来估计浓度 梯子紫外线强度。存在于梯子中的DNA的量是 在文献中可用。
    17. 仔细地消除最高的 可见端粒带和目标质粒骨架与手术刀和  放入分开的1.7ml管中(图2)。


      图2.示例  凝胶用于连接。 pNG4是此的靶标质粒 结扎。切除盒装部分。

    18. 加入1毫升无核酸酶的水凝胶切片,并在4℃下透析过夜,以从琼脂糖中除去盐。
    19. 从管中取出水并置于70°C水浴中直到完全熔化(约5分钟)。
      注意:每分钟一次,以确保琼脂糖完全融化。
    20. 一旦熔化,将管移至37°C水浴。
    21. 在琼脂糖冷却下,将连接酶混合物在单独的1.7ml管中制备(表1)。

      表1.连接反应组分
      组件
      金额
      连接酶缓冲液
      5μl
      T4 DNA连接酶
      1μl(400单位)
      目标质粒骨架
      从凝胶估算出100ng
      端粒
      至50μl
    22. 冷却约5分钟后,加入目标质粒和端粒进行连接 混合物。
      注意:快速工作,琼脂糖不会在移液器尖端固化。
    23. 通过温和的移液混合并使其恢复到室温。
      注意:反应应该在冷却时固化。
    24. 在室温下孵育过夜。
    25. 通过加入1ml水至管中进行透析反应,并在室温孵育 温度15分钟。
      注意:弹簧管直到琼脂糖自由漂浮在水中。
    26. 开始在冰上解冻Stbl4细胞。
      注意:每次反应需要40μl的细胞。
    27. 彻底清除透析水,加入50μl新鲜水 核酸酶免费 水。将管置于70°C水浴中10分钟,或 直到完全融化。
      注意:每分钟轻轻甩管,以确保凝胶完全 融化了。
    28. 向40μlStbl4细胞中加入2μl连接反应 以下电击 厂商推荐设置为 电穿孔机将电穿孔细胞重悬于500毫升S.O.C培养基中 并在30ml培养管中以250rpm在30℃摇动1.5小时。
    29. 将100μl转化制备物置于预热的LB上 含有适当抗生素的板,并在30℃下孵育直至  殖民地出现
      注意:由于温度较低,菌落可能需要2天时间 出现在盘子上。
    30. 筛选具有菌落PCR或菌落杂交的阳性菌落。
      注意:筛选阳性菌落:
      由于端粒的重复性质,不可取 通过在端粒插入位点扩增筛选菌落,as 大多数PCR反应将失败。相反,可以进行菌落PCR 使用正向引物退火5'到插入位点 靶质粒和与5'位点退火的反向引物 pWY82中的端粒重复序列也插入到靶质粒中 如下图3所示。确保良好的底漆是重要的 在进行菌落PCR之前,每个质粒中的功能 引物pWY82R  序列5'GTGGGACCCGAGGGAATC 3'已被常规用作 菌落PCR中的反向引物。


      图3.应选择与插入点相邻的菌落PCR引物,不扩增端粒重复

      或者,可以筛选整个殖民地的平板 通过菌落杂交存在端粒(Sambrook等,1989)。 该方法允许立即快速筛选数百个菌落, 并且非常敏感。寡核苷酸探针用于 杂交由10个单位的端粒重复组成,无论是在  5'或3'方向。最后,建议序列化 端粒重复以确保插入物处于正确的方向。

  2. 检测端粒插入物大小
    一旦获得阳性菌落,筛选菌落的端粒重要是重要的。对于大多数目的,可能需要具有最大端粒插入的菌落进行转化。端粒大小估计最好通过南方转移和杂交(Southern,1975)进行。靶质粒以及质粒pWY82与测试的菌落一起作为对照。
    1. 接种阳性菌落,加上一种pWY82和一种培养物 的原始目标质粒,加入3ml含有的2XYT培养基 适当的抗生素。在30℃下以250rpm摇动培养8小时。
    2. 用100μl的起始培养物接种新鲜的5ml 2XYT培养物 的每个菌落含有适当的抗生素,并在250℃摇匀 在30℃下16-18小时。
      注意:有时有用 接种并生长已经存在的较大(25ml)培养物的菌落 选择。一个等分试样可用于筛选,当克隆已经被使用时 用所需的端粒片段鉴定,剩下的培养物可以  然后用于较大规模的质粒制备。
    3. 使用4 ml的每种培养物,用QIAprep Spin Miniprep提取质粒DNA 套件将质粒提取到40μl温(55℃)核酸酶中 水。
    4. 对1μg每个质粒进行限制性消化  20μl总体积使用将切割为接近端粒的酶  尽可能插入两端,以切除端粒插入物。 另外,用也会切断端粒的酶切割pWY82 插入作参考。
    5. 使用足够大的梳子将标准的1%(w/v)1x TAE琼脂糖凝胶倒入以适应整个限制性消化。
    6. 限制性消化完成后,向每个反应中加入5μl6x负载染料。
    7. 将每个消化物装载到琼脂糖凝胶中,并以100 V运行,直到较低的条带到达凝胶的底部。
    8. 小心地将凝胶转移到含有1 TAE和0.5μg/ml溴化乙锭的培养皿中并染色30分钟。
    9. 用紫外线可视化DNA。
      注意:由于端粒的二级结构,来自限制性摘要的端粒大小的估计将是不可靠的。
    10. 通过Southern转移将DNA转移到硝酸纤维素膜上(Green和Sambrook,2012)。
    11. 将膜与放射性标记的寡核苷酸杂交 与尺寸标准相比较来估计端粒尺寸(绿色和 Sambrook,2012)。
      注意:序列寡核苷酸(TTTAGGG)<10>对于检测端粒的存在是非常敏感的。

  3. 端粒PCR方法生成端粒片段
    可以使用PCR产生不同大小的端粒片段,以用于微染色体生产的共轰击方法。使用如下所示的引物,如图所示进行反应,将产生不同大小的各种端粒聚集体,可通过引物交叉退火产生。这些端粒长度可以通过凝胶电泳显现。可以通过从琼脂糖凝胶中切出所需的端粒尺寸来获得特定长度的端粒DNA。以下步骤将概述如何通过PCR产生端粒片段以及如何利用所获得的DNA。该协议改编自用于制备标记端粒的荧光探针的类似方案(IJdo等人,1991)。
    1. 开发PCR引物
      1. 使用下面的序列进行引物开发。这些将允许生产大型端粒碎片。
      2. 端粒引物:5'(TTTAGGG)10 3'。
      3. 端粒引物反义:5'(CCCTAAA)10 3'
      4. 引物可以稀释到上面列出的微摩尔浓度 下表列出了不同端粒片段的大小

      1.25微摩尔前向端粒引物
      1.25微摩尔的反向端粒引物
      产生更大的片段(见图4)
      25微米前向端粒引物
      25微米的反向端粒引物
      产生较短片段(见图4)

    2. PCR反应组装
      1. 使用校对Taq聚合酶,如LongAmp Taq DNA 来自New England Biolabs的聚合酶,并使用上述浓度 引物。建议至少进行四次反应以获得 在DNA凝胶提取后使用足够的DNA。 PCR反应装置为  如下:
        使用来自NEB的LongAmp Taq 聚合酶的PCR反应装置

        核酸酶自由水
        12微升
        24微升
        5x LongAmp 缓冲区
        4微升
        8微升
        感觉端粒底漆
        0.5微升
        1微升
        反义端粒底漆
        0.5微升
        1微升
        10mM dNTPs
        2微升
        4微升
        LongAmp 聚合酶
        1微升
        2微升

        每次反应20微升 每次反应40微升

        热循环仪协议
        98°C 10秒
        55°C 20秒
        72°C 5秒
        重复5x
        98°C 20秒
        55°C 20秒
        72°C 5秒
        重复5x
        98°C 20秒
        55°C 20秒
        72°C 10秒
        重复5x
        98°C 20秒
        62°C 20秒
        72°C 30秒
        重复5x
        72°C 5分钟

        4°C∞


      注意:建议使用大容量PCR反应  或者准备几个PCR反应以增加量 端粒DNA可供使用。
    3. 使用凝胶电泳来确定产生的片段的大小(图4)。
      注意:
      1. 图4中凝胶图像上染色的DNA的涂片是端粒 不同大小的碎片。图4是40微升PCR的结果 反应。切出与所需尺寸相关的涂片 由梯子指示提供该长度的端粒片段。
      2. 图4中端粒片段的涂片基于大小不同 使用Taq聚合酶以及引物浓度。


        图  凝胶电泳后成像的端粒PCR产物实例:L 表示用于该实验的1kb梯形图。 1.25微米和 25微米表示各自存在的PCR引物的浓度 样品。

      3. 按照凝胶电泳方案(Sambrook和Russell,2001)。
    4. 凝胶提取端粒DNA
      1. 消除与端粒尺寸对应的凝胶片 所需的,并从该凝胶片中提取端粒DNA。例如, 如果需要2 kb端粒片段,切出最接近的凝胶涂片  关系到梯子上的2 kb标记。一般来说,较大的片段  如果目标是截断染色体,则需要端粒,因为 端粒片段越大,插入的机会越大  端粒片段将被端粒封端所识别 机器作为染色体的新端。最小尺寸 该识别所需的端粒片段可能因物种而异  物种(Teo等人,2011)。
      2. 使用向导SV凝胶和PCR 用于从琼脂糖凝胶提取DNA的净化系统。跟着 制造商从琼脂糖凝胶中提取DNA的说明书 片。
        注意:
        1. 大量的DNA会丢失 当进行凝胶提取时。建议使用大容量 PCR反应或多重反应。
        2. 建议 使用无核酸酶的水,例如Nuclease-Free进行洗脱 来自Life Technologies的水,以便进一步使用克隆
    5. 使用提取的DNA
      1. 所得到的端粒DNA片段可用于与转基因的配对。
      2. (Kikkert等人,2005)中所述的端粒DNA的制备可用于共轰击。

  4. 讨论
    在概念上,微染色体可以通过自下而上或自上而下的策略来创建。自下而上的策略将需要人造创建着丝粒,端粒和起始复制序列以创建可行的染色体。目前,复制起始点和着丝粒不能在大多数真核生物中人工制造。自上而下的方法利用现有染色体通过用含有端粒的构建体截断来形成微染色体。端粒介导的截短是产生自上而下的微染色体的有效手段。端粒介导的截短(端粒序列)的重要因素可能难以操作,因为它们的构成是大量的重复序列。这里描述的端粒DNA的创建和使用方法是最有效的。将端粒克隆到载体中后,或者制备端粒用于核苷酸序列,通过转化方案正常进行。组织再生与标准转化相同。截短事件的选择可以使用细胞学技术进行,例如原位杂交(Yu et al。,2007)。这些方法应允许产生所需大小的端粒DNA,如果需要,将其包含入载体中,引入选择的种类中,并选择截短的染色体。

食谱

  1. LB肉汤
    LB肉汤
    对于500ml培养基,将12.5g LB培养基溶于400ml水中 将最终体积加至500ml并高压灭菌20分钟
  2. LB板
    对于500ml培养基,将12.5g LB培养基和6g琼脂溶于400ml水中 将最终体积调至500ml,高压灭菌20分钟 允许冷却至50°C并添加适当的抗生素
    轻轻旋转,并向培养皿添加薄层
    允许冷却,然后倒置并储存在4°C
  3. 2x YT肉汤
    对于500ml培养基,将15.5g 2x YT粉末溶于400ml水中 将最终体积加至500ml并高压灭菌20分钟
  4. 壮观霉素(100mg/ml)
    对于10ml储存液,将1μg壮观霉素加入到10ml锥形管中的10毫升高压灭菌水中, 倒置至完全溶解并用0.2微米过滤器过滤消毒 将等分试样500μl放入1.7 ml管中,储存于-20°C
  5. TAE
    为了使1L 50x TAE储备液加入242g三唑基,14.6g乙二胺四乙酸(EDTA)和57.1ml乙酸至500ml水中并溶解
    将总体积达到1升,用水
    稀释至1x工作油与水

致谢

对该主题的研究得到了植物基因组计划国家科学基金会授权IOS-1339198的支持。

参考文献

  1. Adams,S.P.,Hartman,T.P.,Lim,K.Y.,Chase,M.W.,Bennett,M.D.,Leitch,I.J。和Leitch,A.R。(2001)。 拟南芥型端粒重复序列的丢失和恢复5' - (TTTAGGG)(n)-3 "在开花植物的主要辐射的进化中。"Proc Biol Sci"268(1476):1541-1546。
  2. Birchler,J.A.,Gao,Z.,Sharma,A.,Presting,G.G.and Han,F。(2011)。 植物中着丝粒功能的表观遗传学方面。Curr Opin Plant Biol < em> 14(2):217-222。
  3. Birchler,J.A。和Han,F。(2009)。 玉米着丝粒:结构,功能,表观遗传学。 Annu Rev Genet 43:287-303。
  4. Gaeta,R.T.,Masonbrink,R.E.,Krishnaswamy,L.,Zhao,C.and Birchler,J.A。(2012)。 植物中的合成染色体平台。 Annu Rev Plant Biol 63:307-330。
  5. Green,M.R。和Sambrook,J。(2012)。分子克隆:实验室手册。冷泉港实验室出版社。
  6. Ijdo,J.W.Wells,R.A。,Baldini,A.and Reeders,S.T。(1991)。 使用由PCR产生的端粒重复探针(TTAGGG)n改进的端粒检测。 <核酸研究所19(17):4780。
  7. Kikkert,J.R.,Vidal,J.R。和Reisch,B.I。(2005)。 通过粒子轰击/生物样本稳定转化植物细胞。 286:61-78。
  8. Nelson,A.D.,Lamb,J.C.,Kobrossly,P.S。和Shippen,D.E。(2011)。 在拟南芥中影响端粒介导染色体截短的参数植物细胞 23(6):2263-2272。
  9. Sambrook,J.和Russell,D.W。 (2001)。分子克隆实验室手册。冷泉港实验室出版社。
  10. Sambrook,J.,Fritsch,E.F。,和Maniatis,T。(1989)。分子克隆:实验室手册。冷泉港实验室出版社。
  11. Southern,E.M。(1975)。 通过凝胶电泳分离的DNA片段中的特定序列的检测。生物 98(3):503-517。
  12. Teo,C.H.,Ma,L.,Kapusi,E.,Hensel,G.,Kumlehn,J.,Schubert,I.,Houben,A.and Mette,M.F。(2011)。 诱导拟南芥中端粒染色体截短和截短染色体的稳定性。植物J 68(1):28-39。
  13. Vega,J.M.,Yu,W.,Han,F.,Kato,A.,Peters,E.M.,Zhang,Z.J。和Birchler,J.A。(2008)。 农杆菌介绍玉米(Zea mays)与Cre-lox的转化BIBAC载体中的位点特异性重组盒。(植物分子生物学)66(6):587-598。
  14. Yu,W.,Lamb,J.C.,Han,F.and Birchler,J.A。(2006)。 玉米中端粒介导的染色体截短。 Proc Natl Acad Sci USA 103(46):17331-17336。
  15. Yu,W.,Han,F.,Gao,Z.,Vega,J.M。和Birchler,J.A。(2007)。 玉米中工程微染色体的构建和行为 美国科学院院士(美国) 104(21):8924-8929。
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How to cite this protocol: Graham, N. D., Swyers, N. C., Gaeta, R. T., Zhao, C., Cody, J. P. and Birchler, J. A. (2015). Telomere-mediated Chromosomal Truncation via Agrobacterium tumefaciens or Particle Bombardment to Produce Engineered Minichromosomes in Plants. Bio-protocol 5(18): e1595. DOI: 10.21769/BioProtoc.1595; Full Text



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