Conditional Depletion of STN1 in Mouse Embryonic Fibroblasts

Materials and reagents

Biological materials

1. CreER^T2 C57BL/6 mice harboring the conditional CreER^T2 transgene (Jackson Laboratory, stock 004682)

2. Homozygous Stn1^flox/flox (Stn1^F/F) C57BL/6 mice (generated as described in Nguyen et al. [34])

3. CreER^T2; Stn1^F/F and control Stn1^F/F C57BL/6 mice: generated by crossing with Stn1^F/F with CreER^T2 C57BL/6 mice to generate progeny heterozygous for Stn1^Flox allele and hemizygous for the CreER^T2 genotype. These mice were then bred to homozygous Stn1^F/F mice to derive Stn1 conditional knockout CreER^T2; Stn1^F/F and control Stn1^F/F genotypes as described previously [34].

   
   

1. DMEM/F-12 (Gibco, catalog number: 11320033)

2. Fetal bovine serum (FBS) (Biowest, catalog number: S1620)

3. Antibiotic/antimycotic solution (HyClone, catalog number: SV30079.01)

4. Trypsin-EDTA 0.05% and 0.025% (Gibco, catalog number: 2530062)

5. DMSO (Sigma, catalog number: D-5879)

6. 4-Hydroxytamoxifen (4-OHT) (VWR, catalog number: 89152-604)

7. KOD Hot Start DNA Polymerase kit (Millipore Sigma, catalog number: 710863, solutions provided in the kit)

8. Oligos:

   oIMR5984: 5'-GCTAACCATGTTCATGCCTTC-3' (Cre transgene forward)

   oIMR8744: 5'-CAAATGTTGCTTGTCTGGTG-3' (Cre transgene reverse)

   oIMR8745: 5'-GTCAGTCGAGTGCATAGTTT-3' (Cre internal positive control forward)

   oIMR9074: 5'-AGGCAAATTTTGGTGTACGG-3' (Cre internal positive control reverse)

   Stn1loxPShift-F: 5'-TGTAATCCCAGCGCTCAGGAG-3'

   Stn1loxPShift-R: 5'-GATCTGACAGAGATCTCCTGGCT-3'

   Obfc1 5'wtF2: 5'-GACTCCTTAGCCCCAGATCTCCGTCAT-3'

   Obfc1 5'mtR: 5'-CTTCGTATAGCATACATTATACGAAGTTATGGATCCACCGACT-3'

   Obfc1 5'mtF: 5'-CCACATAGTCGGTGGATCCATAACTTCGT-3'

   Obfc1 5'wtR: 5'-GGGCTAGGGTGTAGCTCAATGGTAGA-3'

   Obfc1 3'wtF: 5'-CTCTGGGGAGACTGTCTCAGATGTTCA-3'

   Obfc1 3'mtR: 5'-GAGGCTCAGGATCCATAACTTCGT-3'

   Obfc1 3'mtF: 5'-GCCAGCCTGGTCTACGTGACAATAACT-3'

   Obfc1 3'wtR: 5'-GGTCATATGGCCTCACCCTCTAAGA-3'

9. Glycerol (Fisher Scientific, catalog number: BP-229-1)

10. Agarose (Sigma-Aldrich, catalog number: A9539-500G)

11. DTT (Sigma-Aldrich, catalog number: D0632-5G)

12. Anti-STN1 antibody (Santa Cruz, catalog number: sc-376450), 1:500 dilution

13. Anti-β-actin (Sigma, catalog number: A2228) 1:60,000 dilution

14. Secondary horseradish peroxidase antibody: TrueBlot anti-mouse IgG HRP conjugated (Rockland, catalog number: 18-8817-30) 1:2,000 dilution

15. SuperSignal West Femto (Thermo Fisher, catalog number: 34095)

16. Hydrogen peroxide (30%, ACS grade) (Ward’s Science, catalog number: 470301)

17. Crystal Violet (Sigma, catalog number: C3886)

18. CHAPS (Fisher Scientific, catalog number: BP571-5)

19. Ethanol (Sigma-Aldrich, catalog number: 459844-4L)

20. Sodium dodecyl sulfate (SDS) (Fisher Scientific, catalog number: BP166-500)

21. Bromophenol Blue (Sigma, catalog number: B-8026)

22. NaOH (Fisher Scientific, catalog number: S318-500)

23. NaCl (Fisher Scientific, catalog number: S271-1)

24. Tris-HCl (Sigma-Aldrich, catalog number: T3253-500G)

25. Na₂HPO₄ (VWR, catalog number: 0404-500G)

26. KH₂PO₄ (J.T. Baker, catalog number: 3246-01)

27. KCl (EMD, catalog number: PX1405-1)

28. Tween-20 (VWR, catalog number: M147-1L)

29. Non-fat dry milk (Nestle, catalog number: 12428935)

    
    

1. 2 mM 4-OHT stock solution (see Recipes)

2. DNA isolation lysis buffer (see Recipes)

3. DNA isolation neutralization buffer (see Recipes)

4. Phosphate-buffered saline (PBS), pH 7.4 (see Recipes)

5. CHAPS lysis buffer (see Recipes)

6. Loading buffer (see Recipes)

7. Crystal violet staining solution (see Recipes)

8. 1% SDS in 70% Ethanol

9. PBS-Tween (PBST)

10. DMEM/F-12 culture medium (see Recipes)

    
    

Recipes

All solutions are prepared with either autoclaved Milli-Q water or DMSO.

1. 2 mM 4-OHT stock solution

   Reagent	Final concentration	Quantity or Volume
   4-OHT	2 mM	7.75 mg
   DMSO	n/a	10 mL
   Total	n/a	10 mL

   Store at -20 °C. Protect from light. For long-term storage, store the solution at -80 °C.

   
   

2. DNA isolation lysis buffer

   Reagent	Final concentration	Quantity or Volume
   NaOH	25 mM	20 mg
   EDTA	0.2 mM	1.17 mg
   H2O	n/a	20 mL
   Total	n/a	20 mL

   Store at room temperature (RT).

   
   

3. DNA isolation neutralization buffer (pH 5.5)

   Reagent	Final concentration	Quantity or Volume
   Tris-HCl	40 mM	69.9 mg
   H2O	n/a	20 mL
   Total	n/a	20 mL

   Store at RT.

   
   

4. PBS

   Reagent	Final concentration	Quantity or Volume
   NaCl	137 mM	8 g
   KCl	2.7 mM	0.2 g
   Na2HPO4	10 mM	1.44 g
   KH2PO4	1.8 mM	0.24 g
   H2O	n/a	1,000 mL
   Total	n/a	1,000 mL

   Store at RT.

   
   

5. CHAPS lysis buffer

   Reagent	Final concentration	Quantity or Volume
   CHAPS 10%	1%	10 mL
   Tris-HCl pH 7.4	30 mM	363.4 mg
   NaCl	150 mM	876.6 mg
   DTT	200 mM	3 g
   H2O	n/a	90 mL
   Total	n/a	100 mL

   Store at -20 °C.

   
   

6. Loading buffer

   Reagent	Final concentration	Quantity or Volume
   SDS 20%	4%	20 mL
   Tris-HCl pH 6.8	100 mM	1.21 g
   Bromophenol Blue	0.2%	0.2 g
   Glycerol	20%	20 mL
   H2O	n/a	60 mL
   Total	n/a	100 mL

   Store at RT.

   
   

7. Crystal violet staining solution

   Reagent	Final concentration	Quantity or Volume
   Crystal Violet	0.5%	2.5 g
   Ethanol	n/a	500 mL
   Total	n/a	500 mL

   Store at RT.

   
   

8. 1% SDS in 70% Ethanol

   Reagent	Final concentration	Quantity or Volume
   SDS 20%	1%	50 mL
   Ethanol	70%	700 mL
   H2O	n/a	250 mL
   Total	n/a	1,000 mL

   Store at RT.

   
   

9. PBST

   Reagent	Final concentration	Quantity or Volume
   NaCl	137 mM	8 g
   KCl	2.7 mM	0.2 g
   Na2HPO4	10 mM	1.44 g
   KH2PO4	1.8 mM	0.24 g
   Tween-20 50%	0.05%	1 mL
   H2O	n/a	999 mL
   Total	n/a	1,000 mL

   Store at RT.

   
   

10. DMEM/F-12 culture medium

    Reagent	Final concentration	Quantity or Volume
    DMEM/F-12	n/a	500 mL
    FBS 100× antibiotic/antimycotic	13% 1×	75 mL 5 mL
    Total	n/a	580 mL

    
    

Laboratory supplies

1. 96-well plates (Genesee Scientific, catalog number: 25-109)

2. 6-well plates (VWR, catalog number: 10062-892)

3. 10 cm tissue culture dishes (Nest Biotechnology, catalog number: 704201)

4. PVDF membrane (MilliporeSigma, catalog number: ISEQ00010)

5. Forceps (Fisher Scientific, catalog number: 08-880)

6. Dissecting scissors (Fisher Scientific, catalog number: 08-940)

7. Scalpel (Fisher Scientific, catalog number: 12-000-164 and 12-000-161)

Equipment

1. Inverted microscope (Fisherbrand, model: 03000013)

2. SDS-PAGE and western blot transfer apparatus (Bio-Rad, model: Mini-PROTEAN Tetra System)

3. Thermocycler (Bio-Rad, model: T100 Thermal Cycler)

4. iBright imaging system (Thermo Fisher, model: CL1000)

5. Plate reader (BioTek, model: Synergy H1)

Software and datasets

1. GraphPad Prism v9.3 (GraphPad, 11/15/2021)

2. ENSEMBL Mouse genome browser 110 (https://useast.ensembl.org/Mus_musculus/Info/Index)

Procedure

1. Design of CRISPR/Cas9 to insert loxP sites

   The mouse Stn1 locus contains 10 exons, with the ATG start codon residing in exon 2 (Figure 1A). When loxP insertion sites were initially designed, the most convenient approach to delete murine Stn1 appeared to be deleting exons 5 and 6, which would create a frameshift and generate a truncated STN1 protein. However, a long non-coding RNA (lncRNA) with unknown function is present in this region. To avoid affecting this lncRNA, we targeted the upstream regions of the Stn1 gene by inserting two loxP sites upstream of the promoter region and downstream of the ATG start codon in exon 2 (Figure 1A). Sequence information and the exact loxP insertion sites are provided in Supplementary File 1. Cre expression resulted in the deletion of the promoter sequence and the translation start site, thereby inhibiting both transcription and translation of the gene (Figure 1A). Deleting the promoter region would also eliminate the possible complications caused by a truncated protein that could be synthesized from a downstream ATG codon. However, we have found that the inducible Stn1 deletion is incomplete, likely due to inefficient recombination caused by the long distance between two loxP sites, resulting in partial knockout or depletion of STN1.

   
   

2. Isolation of mouse embryonic fibroblasts

   1. Set up the breeding pairs of CreER^T2; Stn1^F/F and Stn1^F/F C57BL/6 mice, either male CreER^T2; Stn1^F/F paired with female Stn1^F/F, or male Stn1^F/F paired with female CreER^T2; Stn1^F/F. We have not observed breeding difference.

   2. Euthanize 12–14 days (est.) pregnant mouse with CO₂ followed by cervical dislocation.

      Critical: Before transfer under the cell culture hood, spray mouse down thoroughly with 70% ethanol.

   3. Open the abdominal cavity with sterile scissors. Be careful not to disturb uterine horns.

      Note: Lifting the skin with a sterile forceps before incision and doing the same with the abdominal wall ensures no accidental damage.

      Critical: It is important to keep any hair from being introduced into subsequent steps. Change instruments if necessary.

   4. Extract embryos still within the uterine horns by gently lifting them with forceps and using scissors to cut them free close to the pelvis. Then, place them in a 10 cm dish with ~10 mL of PBS prewarmed to 37 °C.

      Note: If they do not lift out freely, carefully check for locations to cut.

      Critical: It is important not to tear or cut any part of the bowels to avoid bacterial contamination.

   5. Cut open uterus and extract embryos still within amniotic sac; separate embryos by cutting the area between each and remove each embryo while keeping amniotic sac intact.

   6. Open the amniotic sac with forceps and sterile scalpel, remove the embryo, and sever the head above the eye line. Place the severed head in a separate tube on ice for later DNA extraction.

   7. Remove red organs with scalpel and place the remaining tissue in 2 mL of 0.05% trypsin in a 6-well dish.

   8. Gently but thoroughly disrupt tissue with scissors until no piece larger than 1 × 1 × 1 mm remains.

   9. Repeat steps B5–7 for each embryo and place dishes into incubator at 37 °C with 5% CO₂ for 10 min.

      Note: Start incubation within less than 5 min of processing the first embryo, depending on the number of embryos and time required for each. Incubation for separate batches may need to be staggered.

      Critical: Swift processing is recommended to minimize cell death.

   10. Gently pipette cell suspension up and down with a P1000 to further disassociate cells mechanically before they are transferred into 2 mL of DMEM/F-12 culture medium and resuspended again.

   11. After 5 min, transfer the supernatant to a new tube, leaving the larger tissue fragments at the bottom of the tube.

   12. Spin down the new tube at 500× g for 2 min and discard the supernatant; resuspend the cell pellet in 2 mL of fresh DMEM/F-12 culture medium, transfer into a 6-well dish, and place into the incubator overnight.

       Note: Be careful not to mix or pool cells from different embryos.

   13. The next day, replace the medium with fresh medium.

   14. Monitor cells until each well reaches full confluency (1–2 days later) and transfer into a 10 cm dish for expanding and subsequent freezing down of cells.

       
       

3. Validation of genotype

   1. Place a small piece of the tissue from cell isolation (see B6) in 50 μL of DNA isolation lysis buffer (see Recipe 2) and incubate at 98 °C for 1 h.

   2. After incubation, add 50 μL of DNA isolation neutralization buffer (see Recipe 3), pipette the mixture up and down thoroughly to disrupt the rest of the tissue, and transfer into a fresh tube, leaving behind any significant tissue clumps.

      Pause Point: DNA can be stored at -20 °C until needed.

   3. Use 1 μL each of the final mixture to run a PCR reaction with Cre transgene primer pairs and Stn1flox primer pairs.

      1. To determine if animals contain the CreER^T2 transgene, the PCR reactions should contain:

         1 μL lysate

         2.5 μL dNTPs (2.5 mM stock)

         2.5 μL 10× buffer for KOD Hot Start

         1.5 μL oIMR5984 (10 μM)

         1.5 μL oIMR8744 (10 μM)

         1.5 μL oIMR8745 (10 µM)

         1.5 μL oIMR9074 (10 µM)

         0.5 μL KOD

         1.5 μL MgSO₄ (50 mM stock)

         1.25 μL DMSO

         9.75 μL H₂O

         25 μL Total

         Run the PCR under the following thermocycler conditions:

         1. 95 °C for 5 min

         2. 95 °C for 30 s

         3. 55 °C for 30 s

         4. 72 °C for 1 min

         5. Go to 2) for 31 cycles

         6. 72 °C for 5 min

         7. 10 °C infinite hold

      2. To determine if the animal is homozygous or heterozygous of the Stn1 flox allele, the PCR reactions should contain:

         1 μL lysate

         2.5 μL dNTPs (2.5 mM stock)

         2.5 μL 10× buffer for KOD Hot Start

         1.5 μL STN1loxPShift-F (10 μM)

         1.5 μL STN1loxPShift-R (10 μM)

         0.5 μL KOD

         1.5 μL MgSO₄ (50 mM stock)

         1.25 μL DMSO

         13 μL H₂O

         Run the PCR under the following thermocycler conditions:

         1. 95 °C for 5 min

         2. 95 °C for 20 s

         3. 60 °C for 30 s

         4. 72 °C for 40 s

         5. Go to 2) for 34 cycles

         6. 72 °C for 2 min

         7. 10 °C infinite hold

      3. (Optional) To validate if the animal contains the loxP insertions in the correct Stn1 loci, four additional PCR reactions can be performed:

         1 μL lysate

         2.5 μL ldNTPs (2.5 mM stock)

         2.5 μL 10× buffer for KOD Hot Start

         1.5 μL Primer a, c, e, or g (10 μM) (see primer notes below)

         1.5 μL Primer b, d, f, or h (10 μM) (see primer notes below)

         0.5 μL KOD

         1.5 μL MgSO₄ (50 mM stock)

         1.25 μL DMSO

         13 μL H₂O

         Run the PCR under the following thermocycler conditions:

         1. 95 °C for 5 min

         2. 95 °C for 20 s

         3. 60 °C for 30 s

         4. 72 °C for 40 s

         5. Go to 2) for 34 cycles

         6. 72 °C for 2 min

         7. 10 °C infinite hold

            
            

      Notes:

      Primer pair #1: produces a 547 bp product if 5' loxP is present.

      Primer a: Obfc1 5'wtF2, anneals upstream of the 5' loxP site (Figure 1A)

      Primer b: Obfc1 5'mtR, anneals at the 5' loxP site (Figure 1A )

      Primer pair #2: produces a 226 bp product if 5' loxP is present.

      Primer c: Obfc1 5'mtF, anneals at the 5' loxP site (Figure 1A )

      Primer d: Obfc1 5'wtR, anneals downstream of the 5' loxP site (Figure 1A)

      Primer pair #3: produces a 335 bp product if 5' loxP is present.

      Primer e: Obfc1 3'wtF, anneals upstream of the 3' loxP site (Figure 1A)

      Primer f: Obfc1 3'mtR, anneals at the 3' loxP site (Figure 1A )

      Primer pair #4: produces a 550 bp product if 5' loxP is present.

      Primer g: Obfc1 3'mtF, anneals at the 3' loxP site (Figure 1A )

      Primer h: Obfc1 3'wtR, anneals downstream of the 3' loxP site (Figure 1A)

      
      

      
      Figure 1. Genotyping of the Stn1^F/F allele and the CreER^T2 transgene. A. Targeting construct encoding the murine Stn1 gene, which contains 10 exons. Yellow arrowheads designate loxP sites. Arrows designate PCR primer sites. Open box: non-coding exon. The Cre protein is a site-specific DNA recombinase that recombines a pair of loxP sequences. The Cre enzyme binds to the loxP sites and brings the two sites together, catalyzing a recombination event between them, resulting in the excision of the DNA sequence between the two loxP sites. The excised DNA is usually degraded, and the ends of the remaining DNA are ligated together. B. Example of PCR products for detecting the presence of the Stn1^F/F allele. C. Example of PCR products for detecting the CreER^T2 transgene.

      
      

   4. Add 5 μL of 6× loading buffer (see Recipe 6) to each PCR reaction and load 12 μL of the mixture onto a 2.5% agarose electrophoresis gel. Run at 120 V for 1 h.

      Pause Point: PCR reactions can be stored at -20 °C for several days before electrophoresis.

      Expected results and interpretation: The WT allele will produce a 184 bp PCR product, and the recombined flox allele will produce a 218 bp product. If the PCR reaction yields a single 218 bp band, it indicates that the animal is Stn1^F/F; a single 184 bp PCR product means that the animal is Stn1 ^+/+; and a double band of 184 bp and 218 bp means the animal is Stn1^F/+ (Figure 1B). The presence of the CreER^T2 transgene generates a double band (Figure 1C).

      
      

4. Cultivation of MEFs and induced knockout of Stn1

   1. Grow MEF cells in DMEM/F-12 culture medium at 37 °C with 5% CO₂.

   2. Passage cells when they reach near or full confluency at a 1:4 split ratio.

      Note: When cell confluency is too low after passaging, cells take a long time to grow.

   3. To induce Cre expression to deplete Stn1, add 4-OHT into media to a final concentration of 2 μM for 48–96 h.

      
      

5. Immortalization of MEFs

   1. Primary cells are spontaneously immortalized by continuous passaging in DMEM/F-12 medium at 37 °C with 5% CO₂ for several months (20+ passages). Surviving cells that continue to grow and do not senesce can be considered immortalized. Immortalized cells exhibit slightly different morphology (Figure 2).

   2. To induce Cre expression to knockout Stn1, add 4-OHT into media at 2 μM for 48–96 h.

      
      

      
      Figure 2. Cell morphology of primary and immortalized mouse embryonic fibroblasts (MEFs). A. Primary MEF cells tend to grow in aggregates. B. Immortalized MEFs grow more evenly distributed and spread out, cells are contracted and round, and spindles are narrower compared to primary MEFs. Images were taken with Zeiss inverted microscope Observer with a 10× phase contrast lens.

      
      

6. Analysis of protein levels after induction

   1. Extract proteins from 20,000 cells by resuspending cells in 15 μL of CHAPS lysis buffer (see Recipe 5) and incubating on ice for 30 min. Then, spin down at 12,000× g for 15 min at 4 °C and mix the supernatant 1:1 with 2× loading buffer.

   2. Load samples onto 10% SDS-PAGE gel and run at 160 V for 60 min in SDS gel running buffer.

      Note: Exact run time may differ; refer to the location of the loading buffer band and run until it is near the bottom edge of the gel.

   3. Transfer proteins from the gel onto a PVDF membrane in a Bio-Rad transfer chamber at ~100 V and 400 mA for 70 min.

   4. Block membrane in PBST buffer + 5% non-fat milk for 1 h at RT.

   5. Incubate the membrane in primary antibody (anti-STN1 and anti-β-actin) in PBST at 4 °C overnight.

   6. Wash membrane five times in PBST for 10 min each.

   7. Incubate the membrane in secondary HRP antibody in 5% non-fat milk PBST buffer at RT for 1 h.

   8. Wash the membrane five times in PBST for 10 min each.

   9. Apply SuperSignal West Femto mixture diluted 1:5 in ddH₂O to the wet membrane.

   10. Capture western blot signal with iBright CL1000 imaging system. Examples of western blot signals are shown in Figure 3A and 3B.

       
       

       
       Figure 3. Induction of STN1 depletion leads to cell sensitivity to oxidative stress. A. Primary Stn1^F/F and CreER^T2, Stn1^F/F mouse embryonic fibroblast (MEF) clones were treated with 4-OHT (2 μM) for 48 h. STN1 protein expression was analyzed by western blotting. B. Spontaneously immortalized Stn1^F/F and Cre, Stn1^F/F MEF clones were treated with 4-OHT (2 μM) for 48 h. STN1 protein expression was analyzed by western blotting. C. Immortalized MEF clones were seeded to 35 mm dishes and then treated with 4-OHT for 48 h. Cells were then trypsinized and seeded into 96-well plates with 5,000 cells per well. Once cells were attached, hydrogen peroxide was added at the indicated concentration for 24 h. Cell viability was determined by Crystal Violet colorimetric assay. Two independent experiments were performed. Representative results from one experiment are shown. Error bars: S.D.

       
       

7. Testing cell sensitivity to hydrogen peroxide

   1. Grow MEFs in culture, trypsinized, and seed 5,000 cells into each well of a 96-well plate, following induction according to step D3. Maintain 4-OHT in the media. Seed cells in six wells for each sample and each treatment condition.

   2. After cells have settled and have been allowed to attach to dishes for approximately 3 h, add hydrogen peroxide and incubate the plate at 37 °C and 5% CO₂ overnight.

   3. Remove media with a multichannel pipette, wash gently with 200 μL of PBS per well, and remove PBS.

      Note: To avoid accidentally detaching cells, PBS should be added slowly along the side of the wells and in the same manner across the whole plate. Removal of the media and PBS should be done slowly with the plate slightly tilted and the pipette tip in one corner of the wells.

   4. Add 100 μL of Crystal Violet solution (see Recipe 7) to each well and incubate for 15 min at room temperature.

   5. Pour out the Crystal Violet solution and blot firmly against paper towels.

   6. Add ddH₂O with the multichannel pipette, pour out, and firmly blot against paper towels.

   7. Immerse the plate in ddH₂O for 15 min at RT, pour out, and blot against paper towels.

   8. Air dry the plate until completely dry.

      Pause Point: Dried plates can be stored in a dry and dark place at RT overnight if necessary.

   9. Add 200 μL of 1% SDS in 70% ethanol (see Recipe 8) per well and carefully resuspend and mix each well with a multichannel pipette without introducing bubbles.

   10. Measure absorbance at 570 nm with a plate reader. Results are shown in Figure 3C .

Validation of protocol

This protocol or parts of it has been used and validated in the following research article:

Nguyen et al. [34]. Deficiency in mammalian STN1 promotes colon cancer development via inhibiting DNA repair. Science Advances (Figure 4, Figure 6, panels D and E, Figure 7, panel A).

General notes and troubleshooting

Troubleshooting

Problem 1: MEF cells grow slowly.

Possible cause: After a number of passages, primary MEFs senesce.

Solution: Use early passaged MEF cells. It is recommended to freeze down several aliquots of early-passaged primary MEFs for later use.

Problem 2: High background in anti-Stn1 western blot.

Possible cause: Use of regular HRP-conjugated secondary anti-mouse IgG antibody.

Solution: Use the TrueBlot HRP-conjugated anti-mouse secondary antibody.

Problem 3: Immortalized MEF cells lose Stn1 depletion.

Possible cause: We initially used SV40 large T-antigen to immortalize MEFs. However, we have found that MEFs immortalized with this method do not respond to 4-OHT treatment and appear to have lost STN1 depletion. Although the exact cause of this loss is unclear, we suspect that SV40 large T-antigen alters the repair of double-strand breaks, which could influence Cre-loxP recombination efficiency and precision. In addition, large T antigen expression leads to higher mutations and chromosome aberrations, which could impact the sites where Cre recombination acts.

Solution: Use spontaneously immortalized MEFs. Do not use SV40 large T-antigen to immortalize MEFs.

Problem 4: Inefficient STN1 depletion after Cre induction.

Possible cause: Tamoxifen treatment time is not long enough, or tamoxifen concentration is incorrect.

Solution: Cells need to be treated with 4-OHT for at least 48 h to achieve efficient Stn1 depletion. Check the 4-OHT concentration to make sure that it is correct.

Acknowledgments

We thank Duc Dinh Nguyen, Eugene Kim, Thanh Nguyen, and Olga Shiva for technical assistance. This protocol was described and validated in the original research paper: DOI: 10.1126/sciadv.add8023.

Competing interests

Authors declare no financial or non-financial competing interests.

Ethical considerations

Animals were housed and studied in specific pathogen-free animal facilities at Loyola University Chicago Health Sciences campuses. All studies were approved by Institutional Animal Care and Use Committee (IACUC).

References

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Supplementary information

The following supporting information can be downloaded here:

1. Sequence of murine Stn1 (aka OBFC1) locus including the upstream region. Location of the loxP insertion sites and inserted loxP sequence are highlighted. Long red arrows indicate the location of primers used in genotyping.