Key words

1 Introduction

Most mammalian genomes are characterized for the high prevalence of repeated DNA sequences. Among repeated DNA sequences, Transposable Element s (TEs) are repeated DNA sequences that can move within genomes (reviewed in Refs. [1–3]). TEs are very diverse in their structure and abundance depending on the genome that is examined [3]. In humans, between 45 and 70 % of the human genome is made of TEs [4, 5], and active TEs continue to impact our genome [1–3]. Long Interspersed Element class 1 retrotransposons (LINE-1 or L1) are very prevalent sequences in the human genome and up to 21 % of our genome is made of LINE derived sequences [5]. Although most L1 copies are inactive fossils accumulated during human genome evolution, an average human genome contains between 80 and 100 active L1s (termed Retrotransposition Competent L1s or RC-L1s ) [6, 7]. RC-L1s are retrotransposons that move using a copy and paste mechanism termed Target Primed Reverse Transcription (TPRT ) [1–3, 8]. Active RC-L1s are 6-kb in length elements containing a 900-bp long 5′ Untranslated Region (UTR) that contains conserved sense and antisense promoter activities [9–11], two non-overlapping Open Reading Frames (ORF1 and ORF2) and end in a short 3′ UTR region with a weak polyadenylation sequence [12–15]. L1-ORF1p codes for a 40 kDa protein with RNA binding and nucleic acid chaperone activity [16–19]. L1-ORF2p codes for a 150 kDa protein with demonstrated Endonuclease (EN) and Reverse Transcriptase (RT) activities [20–22]. Both ORFs are strictly required for the mobilization of RC-L1, as demonstrated using an engineered L1 retrotransposition assay in cultured cells [14]. Retrotransposition starts with the generation of a full-length polyadenylated L1 mRNA from an active L1 located elsewhere in the genome. The L1 mRNA is exported to the cytoplasm where L1-ORF1p translation takes place by a cap-dependent mechanism [23]. Notably, the L1 mRNA is unusual because is bi-cistronic and studies in cultured cells have revealed that L1-ORF2p is translated by an unconventional termination/reinitiation mechanism [24]. L1-ORF2p translation is very inefficient when compared to L1-ORF1p translation, and although translation of the L1 mRNA might produce hundreds of L1-ORF1p molecules, as little as one L1-ORF2p molecule might be translated from the same L1 mRNA [1–3, 24]. Remarkably, both proteins seem to bind back strongly to the same L1 mRNA used as a template for translation, a concept termed cis-preference [25, 26]. The L1 mRNA and both encoded proteins form a high weight molecular complex termed L1 ribonucleoprotein particle (L1-RNPs) that is supposed to be a retrotransposition intermediate [27]. L1-RNPs can be visualized in cultured cells using epitope tags, and several studies have revealed that L1-RNPs are often located in dense cytoplasmic foci [27–29]. During retrotransposition, L1-RNPs enter the nucleus where, using the intermediate L1 mRNA as a template, a new insertion is generated in a different genomic location, by TPRT. During TPRT, the EN activity of L1-ORF2p is thought to mediate a single strand break on DNA, releasing a free 3′ OH group that is then used by the RT activity of L1-ORF2p as a primer to generate the first cDNA copy of the L1 mRNA attached to the genome of the cell [8]. Second strand cDNA synthesis is thought to occur using a similar mechanism, resulting in the insertion of a new L1 copy in a different genomic place. Because of the L1-EN cleaving mechanism, most de novo L1 insertions are flanked by 2–20 bp Target Site Duplication (TSD ) sequences. However, L1 insertions can also generate small deletions or no duplication of sequence (i.e., blunt insertion), depending on how L1-EN cleaves both strands at the insertion site [30]. Notably, most de novo L1 insertions are 5′ truncated, by an ill-defined mechanism [31]; additionally, some L1 insertions generated inverted/deleted structures by a mechanism termed Twin Priming [32]. These characteristics associated with de novo L1 retrotransposition events are bona fide hallmarks often used to characterize a new L1 insertion in a given genome. Because of their repeated nature and their high prevalence in genomes, identifying new L1 insertions in genomes is a very complicated task equivalent to identify a needle in a haystack.

However, the mechanism of L1 retrotransposition by TPRT allowed the developing of a genetic based L1 retrotransposition assay that is based on the activation of a reporter gene (REP) only after a round of L1-retrotransposition in transfected cultured cells (Fig. 1) [14, 33].

Fig. 1
figure 1

Rationale of the L1-retrotransposition assay. Further details are provided in the text

Full size image

Briefly, in 1996 Moran and colleagues exploit the existence of an intermediate L1 mRNA during L1 retrotransposition to design a reporter gene using an engineered intron with a configuration that allowed expressing a functional reporter product only after a round of bona fide retrotransposition ([14], a method in this book and reviewed in Refs. [34, 35]). Briefly, the modified reporter cassette consists of the ORF that codes for a given reporter gene equipped with an exogenous promoter and polyadenylation sequences cloned in an antisense configuration in the 3′ UTR of an RC-L1. Additionally, the reporter ORF is interrupted by an intron cloned in the same transcriptional orientation as the RC-L1 (Fig. 1). With this configuration, only mRNAs generated from the L1 promoter can remove the intron by cis-splicing and the resulting chimeric L1 mRNA go trough a round of L1 retrotransposition, resulting in the insertion of a reporter gene lacking the intron and thus allowing efficient expression of the reporter.

The retrotransposition assay has been instrumental to increase our knowledge of L1 biology. The original assay used the sequence of the neomycin phosphotransferase gene as a reporter (mneoI cassette [14, 33]) and cells harboring a new retrotransposition event could be selected in culture using the mammalian antibiotic neomycin or G418 (Fig. 1). Since then, a number of reporter ORFs have been used to developed different retrotransposition markers (also covered in this Book and reviewed extensively elsewhere). Notably, using the mneoI retrotransposition indicator cassette , new L1 insertions in the genome of cultured cells are tag with a unique spliced mneoI sequence, which further allowed to characterize L1 insertions using conventional library construction/screening or inverse PCR methods [14, 26, 36]. Indeed, the characterization of de novo engineered L1 insertions using this assay in cultured cells has allowed to demonstrated how L1 can delete genomic DNA upon insertion, can mediate major alterations at the insertion site, can retrotranspose in mammalian neuronal cells, how L1 can insert by a new mechanism of insertion independent of its endonuclease, etc. [13, 30, 37–42].

In 2002, Gilbert and colleagues developed a modified mneoI-based retrotransposition indicator cassette that allows the recovery of engineered L1 insertions as autonomously replicating plasmids in bacteria [39] (Fig. 2).

Fig. 2
figure 2

Structure of the engineered L1-recovery plasmid. Shown is a scheme of a RC-L1 containing a recovery retrotransposition indicator cassette (termed K7i)

Full size image

Briefly, as the codifying sequence NEO confers G418 resistance in mammalian cells and kanamycin resistance in bacteria, Gilbert and colleagues modified the mneoI retrotransposition indicator cassette and included a prokaryotic promoter (EM7) and a Shine–Dalgarno sequence upstream the initiating AUG codon of NEO. Additionally, Gilbert and colleagues added a bacterial origin of replication (ColE1) downstream of the modified mneoI cassette and upstream of the L1 polyadenylation sequence. With this configuration, a new retrotransposition event will deliver into the genome of cells a spliced modified mneoI and a colE1 sequence, which is the basis of the generation of a prokaryotic plasmid (Fig. 2).

Thus, cells are first transfected with a plasmid containing an RC-L1 tagged with the recovery indicator cassette and cells containing a new retrotransposition evens can be selected with G418 . Next, foci are expanded (either as single colonies or pools) and cells allowed to grow to obtain enough cells to isolate at least 20 μg of genomic DNA (gDNA). The gDNA of a colony is next digested with a restriction enzyme that cleaves frequently in the genome of the host cells (for human cells SspI, HindIII, BglII, etc.) and that ideally does not cut within the sequence of the transfected plasmid (or if it does, an enzyme that cleaves as closer as possible to nucleotide 1 of the transfected L1). The digested DNA is next ligated in very diluted conditions to favor intramolecular ligation events, resulting in the generation of a prokaryotic plasmid structure (Fig. 3).

Fig. 3
figure 3

Rationale of the recovery assay. Details are included in the text. R.S., restriction site

Full size image

This mix of ligated DNAs is next transformed to ultracompetent E. coli cells, and after recovery of transformed cells, the engineered L1 insertion plasmid is selected using kanamycin in bacteria (Fig. 3). These colonies will have replicated the engineered retrotransposed product plus flanking genomic DNA as a plasmid that can be extracted and purified using conventional miniprep purification of DNA. Finally, these plasmids can be sequenced using Sanger methodology and the characteristics associated with new L1 retrotransposition events inferred at the nucleotide level (Fig. 3 and see [39]). In this chapter, we cover the recovery process, which is essentially the assay described by Gilbert and colleagues in 2002 with minor modifications that increase the efficiency of the recovery process. Notably, alternative methods based on similar methodologies have been also described [43], although they are not covered in this chapter.

2 Materials

2.1 Retrotransposition Assay and Establishment of Cell Lines

  1. 1.

    Cell culture medium for the cell line to be used (see Note 1 ).

  2. 2.

    10 cm cell culture dishes.

  3. 3.

    Appropriate transfection reagent (Fugene 6 (Promega), Xtreme (Roche), Lipofectamine (Invitrogen), etc.; see Note 1 ).

  4. 4.

    50 mg/ml geneticin (G418 ).

  5. 5.

    Trypsin 0.05 or 0.25 % (see Note 1 ).

  6. 6.

    70 % ethanol.

  7. 7.

    0.2 × 0.2 cm 3MM sterilized chromatography paper (Whatman).

  8. 8.

    Sterile small forceps.

  9. 9.

    24-well tissue culture plates.

  10. 10.

    T75 tissue culture flasks.

  11. 11.

    Cryopreservation tubes and cryopreservation media (see Note 1 ).

  12. 12.

    1× PBS.

  13. 13.

    An engineered L1 construct containing the recovery retrotransposition indicator cassette (as published by the Moran lab) [39–41]. The plasmid might be purified from bacteria using a Midi or Maxiprep purification kit (from local providers). The plasmid used for cell transfection should be in a supercoiled form (80 % or higher).

2.2 Genomic DNA Extraction

  1. 1.

    Blood and Cell Culture DNA midi kit (Qiagen) or a similar kit that provided high quality genomic DNA.

  2. 2.

    Cell lysis buffer: 10 mM Tris–HCl, pH 8.2, 10 mM EDTA, 200 mM NaCl, 0.5 % (w/v) SDS. To prepare 100 ml of lysis buffer: 0.12114 g Tris–HCl; 0.29225 g EDTA; 1.17 g NaCl; 0.5 g SDS (powder) Adjust pH with HCl.

  3. 3.

    100 % ethanol.

  4. 4.

    20 mg/ml proteinase K.

  5. 5.

    25:24:1 phenol–chloroform–isoamyl alcohol.

  6. 6.

    TE buffer, pH 8.

  7. 7.

    Sterilized 1.7 ml microfuge tubes.

  8. 8.

    70 % ethanol.

2.3 Recue of Retrotransposition Events

  1. 1.

    Hind III and SspI enzymes (New England Biolabs).

  2. 2.

    10× Buffer 2 and SspI 10× buffer (New England Biolabs).

  3. 3.

    100× BSA (New England Biolabs).

  4. 4.

    T4 DNA ligase (New England Biolabs).

  5. 5.

    10× T4 DNA ligase buffer (New England Biolabs) (see Note 2 ).

  6. 6.

    Ultra 0.5 ml Centrifugal Filters Ultracel—100 K (Amicon).

  7. 7.

    Ultracompetent XL1-gold or XL1 Blue bacteria (see Note 3 ).

  8. 8.

    15 ml Falcon tubes.

  9. 9.

    Liquid LB culture medium.

  10. 10.

    140 mm bacterial plates.

  11. 11.

    25 mg/ml kanamycin.

  12. 12.

    Wizard Plus SV Miniprep DNA purification system (Promega) or a similar kit from local suppliers.

  13. 13.

    EcoRI (New England Biolabs).

  14. 14.

    10× EcoRI buffer (New England Biolabs).

  15. 15.

    5 ml sterilized bacteria culture tubes.

2.4 Sequencing

  1. 1.

    Primers to sequence recovery plasmids (see Table 1).

    Table 1 Sequence of primers commonly used to characterize L1 insertions using the recovery method
    Full size table

3 Methods

General note: standard good practices for handling cells should be used, including the use of a certified laminar flow biosafety hood and sterile materials and techniques for any reagent used. Note that mammalian cells are very sensitive to DNA transfection when infected by Mycoplasma spp. We routinely test for Mycoplasma spp. once a month. We also verified the nature of cell lines using short tandem repeat (STR ) analyses to ensure cross-contamination with other cell lines.

3.1 Retrotrans-position Assay

  1. 1.

    Trypsinize, count, and seed an appropriate number of cultured cells in a sterile 10 cm cell culture plate (see Notes 1 and 4 ).

  2. 2.

    Transfect cells with the L1 recovery plasmid (see Notes 1 and 5 ) 12–14 h after seeding.

  3. 3.

    After 12 h, change media in the 10 cm plates.

  4. 4.

    72 h post-transfection, replace medium and add medium supplemented with G418 (see Note 6 ).

  5. 5.

    Allow G418 to select G418-resistant foci; the process take a minimum of 10 days but can change between cell lines (see Notes 1 and 6 ). Change the media every other day. See also Note 7 .

3.2 Establishment of G418-Resistant Cell Lines (See Note 8 )

  1. 1.

    Take 10–20 small sterilized 0.2 × 0.2 cm 3MM paper squares and soak them in Trypsin using a sterile 10 cm tissue culture plate (see Note 1 ).

  2. 2.

    Sterilize forceps by immersion in 70 % ethanol. Air-dry forceps.

  3. 3.

    Aided with the sterilized forceps, put a trypsin soaked 0.2 × 0.2 cm 3MM square paper on top of a colony. Incubate at RT for 1–3 min.

  4. 4.

    Using the forceps, put the piece of 3MM paper in a well of a 24-well plate containing 1 ml of complete media. Shake the plate right/left and up/down 2–3 times and incubate cells overnight in an incubator (see Note 9 ).

  5. 5.

    Repeat steps 2–4 as many times as required.

  6. 6.

    Next day, remove papers using a vacuum sucker device and add 1 ml of complete cell culture medium to each well.

  7. 7.

    Expand G-418 foci following standard procedures until obtaining a T-75 full flask of cells (95 % confluent).

  8. 8.

    From the T-75 flask , trypsinize cells and freeze at least one vial of cells using an appropriate method for the cell line of choice (see Note 1 ). Collect the remaining cells using a Falcon 15 ml tube by centrifugation at 1500 × g for 5 min at 4 °C. Remove supernatant and store pellets at −80 °C.

3.3 Genomic DNA Isolation for the Rescue of Engineered LINE-1 Retrotransposition Events (See Note 10 )

  1. 1.

    Extract genomic DNA from cell lines (see Note 11 ). The starting amount of cells required to obtain enough genomic DNA depends on the method used to isolate DNA (see Note 12 ).

  2. 2.

    If using a commercial DNA extraction kit, follow the manufacturer’s instructions. Jump to step 4.

  3. 3.

    If using the homemade protocol, follow these steps:

    1. (a)

      Thaw frozen pellets on ice.

    2. (b)

      Resuspend the cell pellet in 0.5 ml lysis buffer.

    3. (c)

      Add 5 μl of 20 mg/ml proteinase k (see Note 13 ).

    4. (d)

      Incubate for 3 h at 56 °C. If necessary, the digestion can be done overnight at 56 °C (see Note 13 ).

    5. (e)

      Transfer the lysates into clean 1.7 ml tubes or 2 ml tubes depending on the volume of your lysate.

    6. (f)

      Add 1 volume of phenol–chloroform–isoamyl alcohol and shake tubes vigorously.

    7. (g)

      Centrifuge for 5 min at max speed at room temperature (RT) using a microfuge.

    8. (h)

      Keep the aqueous phase (top part) and transfer it to a new 1.7 ml tube (see Note 14 ).

    9. (i)

      Add 2 volumes of 100 % ethanol and place tubes at −20 °C for at least 3–4 h or overnight.

    10. (j)

      Centrifuge tubes at max speed at 4 °C for 15 min. Discard the supernatant.

    11. (k)

      Wash the pellet with 500 μl of 70 % ethanol and centrifuge at max speed for 10 min at 4 °C. Discard supernatant.

    12. (l)

      Air-dry pellets (see Note 15 ) but do not over-dry.

    13. (m)

      Add 200 μl of TE buffer, pH 8.0 and dissolve the pellet. If the pellet is resistant to solution, heat the tube at 56 °C for 1 h with strong agitation. If necessary, incubate it longer.

  4. 4.

    Quantify the concentration of your genomic DNA sample. We recommend using an spectrophotometer as measurements are more accurate that when using a NanoDrop or similar devices.

3.4 The Recovery Protocol (See Note 10 )

Below, we described the recovery process divided in days.

Day 1

  1. 1.

    Genomic DNA digestion. Digest 8 μg of genomic DNA with either HindIII or SspI (see Note 16 ) at 37 °C overnight as follows (see Note 17 ):

    • 5 μl of HindIII or SspI.

    • 10 μl of buffer number 2 or 10 μl of SspI buffer.

    • 1 μl of BSA 100×.

    • 8 μg of genomic DNA.

    • Complete with DNA-free ddH2O up to 100 μl (see Notes 10 and 18 ).

Day 2

  1. 1.

    In the morning, add 1 μl of either HindIII or SspI and incubate at 37 °C for 2 h. Heat at 65 °C to inactive HindIII or SspI enzymes for 25 min.

  2. 2.

    Set up ligation. Add the following reagents to the 100 μl digested DNA sample:

    • 8 μl of T4 DNA ligase.

    • 40 μl of 10× T4 DNA ligase buffer.

    • 351 μl of ddH2O (see Notes 10 and 18 ).

  3. 3.

    Incubate overnight at 16 °C for HindIII digested DNAs and at 25 °C for SspI digested DNAs (see Note 19 ).

Day 3

  1. 1.

    In the morning, add 1 μl of T4 DNA ligase and incubate tubes for at least 4 h at RT.

  2. 2.

    Concentrate DNAs as follows:

    • Transfer each ligation into a clean Amicon.

    • Centrifuge at 5000 × g for 5 min at RT. Discard the flow-through.

    • Add 450 μl of DNA-free ddH2O (see Notes 10 and 18 ).

    • Centrifuge at 5000 × g for 5 min at RT. Note that a small volume of liquid will remain in the Amicon. Do not discard it.

    • Place the Amicon upside down and put it inside a new 1.7 ml tube (see Note 20 ).

    • Short spin. The volume of the concentrated DNA should be around 50–60 μl. If larger, recentrifuge at 5000 × g for 5 min at RT.

  3. 3

    Bacteria transformation.

    • Slowly thaw on ice a 500 μl aliquot of XL1 Blue ultracompetent cells (see Note 21 ).

    • Label 15 ml tubes and cool them on ice. Also, prewarm an aliquot of sterile LB medium at 37 °C (see Note 22 ).

    • Add 1/3 of the concentrated DNA solution (15–20 μl) to the thawed bacteria. Transfer the mix slowly to a pre-cooled 15 ml tube.

    • Incubate on ice for 25–30 min.

    • Heat shock at 42 °C for 38 s. CRITICAL: Do not exceed this time.

    • Incubate on ice for 2 min.

    • Add 1 ml of prewarmed LB (37 °C). Incubate the mix overnight at RT on a shaker but do not exceed 600 rpm (see Note 23 and see Fig. 4).

      Fig. 4
      figure 4

      A system to shake transformed bacteria. See text for further details

      Full size image

Day 4

  1. 1.

    Pellet bacteria by centrifugation at 500 × g for 8 min at RT.

  2. 2.

    Remove supernatant from tubes but keep around 200–300 μl.

  3. 3.

    Resuspend the bacterial suspension carefully with a pipette.

  4. 4.

    Plate the suspension of bacteria in 140 mm LB-Agar plates containing 25 μg/ml kanamycin . Incubate at 37 °C overnight (see Note 24 ).

Day 5

  1. 1.

    After 24 h, colonies should be already visible. In the afternoon, pick individual colonies using an sterilized tip and inoculate a tube containing 2–4 ml of LB with 25 μg/ml kanamycin . Plates with colonies can be also stored at 4 °C for a week.

  2. 2.

    Incubate tubes overnight at 37 °C on a shaker.

Day 6

  1. 1.

    Extract plasmid DNA from bacteria cultures using a miniprep kit. Follow manufacturer’s instructions.

  2. 2.

    Digest a 5 μl aliquot of each recovered plasmid with EcoRI using a standard protocol (see Note 25 ).

  3. 3.

    Resolve digestions on 1–1.5 % agarose gels and identify particular restriction patterns (Fig. 5 and Note 25 ).

    Fig. 5
    figure 5

    Representative results from the recovery assay. Shown are results of plasmids isolated from two G418 -resistant foci and digested with EcoRI. In cell line #2, two patterns can be observed which upon sequencing revealed that this foci contained at least two engineered L1 insertions

    Full size image

3.5 Sequencing Recovered Plasmids

  1. 1.

    Select the plasmids with a unique restriction pattern and sequence them with oligonucleotides Neo 210AS and Reco3 (see Table 1). See Note 26 .

4 Notes

  1. 1.

    The recovery assay can be used in any cell line that support engineered L1 retrotransposition . The media, the transfection conditions, freezing conditions and other general methods must be optimized for each cell line.

  2. 2.

    To avoid degradation of ATP in cycles of thawing/freezing, aliquot T4DNA ligase 10× buffer in 100 μl aliquots.

  3. 3.

    XL1-gold or XL1-blue ultracompetent bacteria can be prepared in house or purchased from local vendors. It is critical that bacteria are ultracompetent.

  4. 4.

    In our hands, we have been able to recover insertions from any cellular type than can support engineered L1 retrotransposition . The number of cells that need to be plated for each cell line might be determined experimentally. The goal of the assay is to have enough colonies dispersed in the 10 cm plate after G418 selection. For HeLa cells, we routinely seed 4–8 × 104 cells/10 cm plate.

  5. 5.

    Similarly, the transfection method or reagent that can be used depends on the cellular type used. We routinely use transfection reagents such as Fugene 6 or Xtreme 9, as both reagents allows transfecting cells plated at a low cellular density without elevated toxicity. For each transfection reagent, follow manufacturer’s instructions.

  6. 6.

    The concentration of G418 used for selection should be empirically determined for each cell line. For HeLa cells, we routinely use 400 μg/ml.

  7. 7.

    It is informative to use an internal control in the retrotransposition assay, by using alleles of the engineered L1 plasmids containing missense mutations in domains involved in retrotransposition (EN and/or RT missense mutants).

  8. 8.

    To avoid cross-contamination in the establishment of G418 resistant cell lines, G418-foci should be distinct in plates, separated by at least 0.5 cm from nearby foci.

  9. 9.

    The well size depends on the ability of cells to grow at a low cellular density. Some cell lines can grow very well starting from few cells in a large culture dish while others are slower/incapable of generating foci. Thus, the well size will vary among cell lines and it must be empirically determined.

  10. 10.

    The recovery process is very sensitive to cross-contamination with other plasmids that use kanamycin as selection. Unfortunately, these plasmids are very common in most labs and special attention should be paid to avoid cross-contamination. Use pipettes and a microfuge devoted only for the recovery process; alternatively, clean all used equipment with diluted bleach and 70 % ethanol.

  11. 11.

    DNA extraction can be performed either using a specific kit (i.e., Qiagen) or using a homemade protocol. In our hands, both protocols are equally efficient but, of course, the costs associated with the homemade protocol are lower.

  12. 12.

    If using a kit, just follow the manufacturer’s instructions about required number of cells. If using the homemade protocol collect ≈5 × 106 (i.e., half of a T75 flask of cells at a 95 % confluence).

  13. 13.

    After 3 h, check the transparency of the lysate. If the lysate is dense/cloudy and it is difficult to pipette up and down, leave it overnight but re-add 0.5 ml of lysis buffer and 5 μl of 20 mg/ml proteinase k.

  14. 14.

    Two phases form: the top aqueous phase (contains the genomic DNA) and the lower phase (organic phase, undigested cellular components will be there). Be careful when collecting the top phase pipetting since it is very easy to take part of the organic phase. Pipette very carefully and do not try to recover the whole upper phase. 2/3 or 3/4 at the most might be sufficient to extract enough but purified genomic DNA.

  15. 15.

    In order to quickly dry pellets, if possible, leave tubes open inside a fuming hood to efficiently remove the remaining ethanol.

  16. 16.

    In our lab, we routinely conduct independent HindIII and SspI digestions to increase the efficiency of the recovery process.

  17. 17.

    We recommend starting in the afternoon (16.00 PM).

  18. 18.

    Be especially careful to avoid cross-contamination with kanamycin resistant-containing plasmids. We recommend acquire commercial ddH2O purified water and prepare 5 ml aliquots.

  19. 19.

    HindIII produces overhang ends while SspI generates blunt ends.

  20. 20.

    We routinely use the collection tubes that are provided with the Amicon tubes.

  21. 21.

    It is important to use XL1-Blue cells, as they can replicate methylated genomic DNAs. Other strains of E. coli can be used, but make sure they are ultracompetent and that they can replicate methylated mammalian DNAs.

  22. 22.

    Avoid any cross-contamination with kanamycin resistant-containing plasmids. We recommend acquire commercial LB and prepare 5 ml aliquots.

  23. 23.

    In our lab, we use a very simple system: a polyspam rack attached to a Thermomixer with tape. A conventional shaker can also be used, but make sure temperature is not higher than 25 °C.

  24. 24.

    We recommend to seed transformations either late in the morning or soon in the afternoon, since recovery colonies need about 24 h to be visible by eye.

  25. 25.

    An EcoRI digestion will allow you to distinguish among different insertions. In fact, sequence those that do not have the same restriction pattern upon EcoRI digestion as they might be different insertions that occurred in the same colony.

  26. 26.

    Sequencing with Reco3 will reach the poly A tail of the L1 insertion. If the polyA tail it is not too long, the inferred DNA sequence might be sufficient to identify the 3′ region of the insertion. If Neo 210AS does not allow you to reach the genomic region at the 5′ end of the insertion, keep sequencing upstream with the rest of the primers that anneal on the LINE-1 sequence (Table 1).