TITLE: Basic Protocol 1: Generation of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 progenitor cells
AUTHORS: Kizhakke Mattada Sathyan, Thomas G. Scott, Michael J. Guertin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The first procedure for implementing the ARF-AID system is to establish ARF-TIR1 progenitor cells, as shown in Figure 2A. All the plasmids for integrating ARF-TIR1 into the AAVS1 locus in human cells are available from Addgene. The choice of transfection method varies depending on the cell type; lipofectamine 3000 works efficiently for HEK293T cells.</div></div>

[STEPS]
?. [Cotransfection of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 and AAVS1 sgRNA]
Remove media from the HEK293T cells and wash with PBS. Add  trypsin to the plate. Incubate the cells for  to , rinse and collect cells with  of media.
0.5 ml
10 ml
?. [Cotransfection of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 and AAVS1 sgRNA]
Remove trypsin by centrifugation at  for  using a swinging bucket centrifuge, remove supernatant, and resuspend the cells in  fresh media.
Centrifuge: 500 34
10 ml
?. [Cotransfection of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 and AAVS1 sgRNA]
Seed 2.0-3 x105 cells per well of a six-well plate to get 30 to 40% confluent cells the next day.
Cotransfect 1 µg each of AAVS1 sgRNA (Addgene # 126582) and eGFPARF-P2A-TIR1 (Addgene # 129668) or ARF-HA-P2A-TIR1 (Addgene # 126582) plasmids with lipofectamine 3000 reagents. Transfection reagents or methods of introducing foreign DNA into cells will vary by cell line. For HEK-293T cells we recommend a ratio of 2.5 µl of lipofectamine for every µg of DNA. As a negative control, transfect the parental vector pX458 with the repair plasmid and include one well as non-transfected control.
?. [Cotransfection of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 and AAVS1 sgRNA]
Add  Opti-MEM I reduced serum media in two 1.5 ml tubes for each transfection; one tube is labeled with the description of the DNA sample and the other is labeled as Lipofectamine. Add  Lipofectamine 3000 reagent to the tube labeled Lipofectamine. Add  p3000 reagent to the tube labeled DNA sample.
125 µl
5 µl
5 µl
?. [Cotransfection of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 and AAVS1 sgRNA]
Add  each of AAVS1 sgRNA (Addgene # 126582) and eGFP-ARF-P2A-TIR1 (Addgene # 129668) or ARF-HA-P2A-TIR1 (Addgene # 126580) plasmids into the p3000 reagent mixture and mix by pipetting up and down three times. Transfect parental vector pX458 with eGFP-ARF-P2A-TIR1 or ARF-HA-P2ATIR1 as negative control.
1 µg
If the transfection efficiency is low, increase the amount of Lipofectamine 3000 and p3000 reagents to 7.5 µl.
?. [Cotransfection of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 and AAVS1 sgRNA]
Transfer the lipofectamine mixture (step 4) to the DNA/p3000 mixture (step 5) dropwise slowly. This tube contains ~ of transfection reagents, DNA and Opti-MEM. Mix by pipetting up and down three times with the same pipette tip.
260 µl
?. [Cotransfection of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 and AAVS1 sgRNA]
Incubate at  for  and transfer the DNA complex into the cells dropwise in a serpentine pattern to uniformly distribute the reagent across the plate.
0 Room temperature
?. [Cotransfection of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 and AAVS1 sgRNA]
Rock the plate sideways four times and front to back four times to distribute the reagent even over the cells--do not swirl the plate. Place cells in the incubator.
?. [Cotransfection of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 and AAVS1 sgRNA]
Twent-four hours after transfection, replace the media with fresh media
?. [Cotransfection of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 and AAVS1 sgRNA]
Forty-eight hours after transfection expand each well of the six-well plate into a 10 cm plate with  media as described in steps 1 and 2 .
10 ml
Expanding cells into 10 cm plates spreads colonies ensures that the cells are sufficiently sparse to allow clonal colonies to form in isolation. Transfect multiple wells to ensure that a sufficient number of colonies survive.
?. [Cotransfection of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 and AAVS1 sgRNA]
Seventy-two hours after transfection, begin selection by adding  puromycin. Antibiotic selection concentration varies between cell types. We recommend plotting a titration vs. cell viability curve to determine the lowest concentration at which nearly all cells die within 5 days.
If the number of colonies are low, begin selection after 120 hours, as opposed to 72. The addition of conditioned media (8:2 ratio of fresh media to filtered cultured media from the same cell type) increases the survival of the clones.
?. [Cotransfection of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 and AAVS1 sgRNA]
After three days of selection replace with fresh media containing puromycin () and continue selection for two days.
?. [Cotransfection of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 and AAVS1 sgRNA]
(optional) After five total days if there are many remaining cells in the negative control plate, then replace with fresh media containing puromycin and continue selection for two days for a total of seven days under selection.
?. [Cotransfection of eGFP-ARF-P2A-TIR1 or ARF-HA-P2A-TIR1 and AAVS1 sgRNA]
After five to seven days under selection all the cells in the untransfected control well should be dead. Remove selection media and expand cells without selection.
Allow colonies to grow and expand. Usually, it takes 2 to 3 weeks for colonies to appear after beginning the puromycin selection (Figure 2A&5), but the time frame is variable and dependent upon the cell line. Check the plate under a microscope and scan for colonies that appear after two weeks. The presence of a substantially higher number of colonies in the transfected plates compared to the control plasmid-transfected or untransfected plates suggests successful integration of the plasmid. The control sgRNA plasmid transfected plates often contain some colonies, but many fewer.
?. [Picking colonies]
Mark individual colonies on the bottom of the plate using a marker. Check eGFPARF-P2A-TIR1 cells using a fluorescent microscope to identify GFP-positive colonies. Select colonies in which all the cells in the colony exhibit uniform nuclear expression of GFP. If the plasmid is modified to localize ARF into cytoplasm or uniformly throughout the cell, then mark those with appropriate GFP distribution. In the case of the ARF-HA-P2A-TIR1 transfected cells select all colonies.
?. [Picking colonies]
Carefully pick individual colonies by taking  trypsin solution using a P20 pipette set to 20 µl. Alternatively, use cloning cylinders to pick individual colonies.
10 µl
For hand picking colonies without the use of cloning cylinders: 1) hold the plate at a 45-degree angle to pool the media away from the colonies; 2) use a P20 pipette set to 20 µl and aspirate ~ of trypsin solution into the pipette tip; 3) dispense a few microliters of the trypsin solution onto the colony such that a small droplet is formed between the plate and the pipette tip, taking care not to dispense so much that the droplet rolls down the plate; 4) scrape the colony with the pipette tip while the trypsin media remains as a bridge between the plate and the tip; and 5) once the colony is dislodged, aspirate the cells into the tip and transfer of the entire colony to the 96 well plate. Resuspend the colonies in   media and transfer into 24 well plates containing 1 ml media per well.Using cloning cylinders: 1) dispense silicone grease onto a glass petri dish and autoclave it along with a pair of forceps; 2) aspirate media from the plate and wash with PBS; 3) hold the plate at a 45-degree angle to pool the PBS away from the colonies; 4) use forceps to pick up a cloning cylinder, dip the thicker edge into silicone grease, and place it over the colony; 5) add   of trypsin solution and incubate at   until cells begin to detach; 6) resuspend the colony in   media and transfer into a 24 well plate containing 1 ml media per well.
?. [Picking colonies]
When the cells reach confluency continue to the next step.
?. [Expanding cells, screening for genomic integration of the ARF-TIR1, and Western blotting]
Collect cells by pipetting up and down and transfer  into a new plate containing media to continue passaging the cells. Transfer the remaining ~ into a 1.5 ml tube.
100 µl
0.9 ml
The plate allows the expansion of the positive colonies. HEK293T cells attach loosely to the plate, so pipetting is enough to dislodge the cells. For other cell lines, collect cells by trypsinization.
?. [Expanding cells, screening for genomic integration of the ARF-TIR1, and Western blotting]
Aliquot  of the  within the eppendorf tube into a fresh tube for genomic DNA isolation and PCR. Place cells immediately .
100 µl
0.9 ml
on ice
?. [Expanding cells, screening for genomic integration of the ARF-TIR1, and Western blotting]
Centrifuge the remaining  of cells at  for  using a fixed angle rotor table top centrifuge and remove media.
0.8 ml
Centrifuge: 6000 34
?. [Expanding cells, screening for genomic integration of the ARF-TIR1, and Western blotting]
Add  2x SDS sample buffer (Laemmli buffer) and mix thoroughly by pipetting up and down to generate cell lysate for Western blotting.
100 µl
?. [Expanding cells, screening for genomic integration of the ARF-TIR1, and Western blotting]
Heat the lysate at  for  on a heating block and vortex for . Place back on the heating block for another . Briefly centrifuge the samples at  and store at .
95 °C
Centrifuge: 5000 34
-20 °C
?. [Screening genomic integration at the AAVS1 locus]
To screen the integration of ARF-TIR1 at the AAVS1 locus, we used genomic PCR using primers that amplify the integrated plasmid DNA.
?. [Genomic DNA isolation]
Centrifuge cells from step 21 at  for  using a fixed angle rotor table top centrifuge, remove media, and either flash freeze or proceed immediately with gDNA isolation.
Centrifuge: 6000 34
Use any genomic DNA isolation kit or conventional phenol-chloroform isolation of DNA to isolate DNA from the cells. Here we used the Qiagen genomic DNA isolation kit. The method described here is adapted from the kit manual with minor changes (DNeasy Blood and Tissue Kit, 69504).
?. [Genomic DNA isolation]
Resuspend the cells in  PBS and add  proteinase K.
200 µl
20 µl
?. [Genomic DNA isolation]
Lyse cells by adding  Buffer AL and mix thoroughly by vortexing to form a homogenous lysate.
200 µl
?. [Genomic DNA isolation]
Incubate samples at  for .
56 °C
?. [Genomic DNA isolation]
Add  100% ethanol and vortex.
200 µl
?. [Genomic DNA isolation]
Transfer the lysate into a DNeasy Mini spin column placed in a 2 ml collection tube.
?. [Genomic DNA isolation]
Spin at max speed in a tabletop centrifuge ( - ) for  and discard the flow-through.
Centrifuge: 10000 34
Centrifuge: 17000 34
?. [Genomic DNA isolation]
Place the spin column back into the collection tube and add  Buffer AW1. Spin at  -  and discard the flow-through.
500 µl
Centrifuge: 10000 34
Centrifuge: 17000 34
?. [Genomic DNA isolation]
Place the spin column back into the collection tube and wash by adding  Buffer AW2 and centrifuging at  -  for .
500 µl
Centrifuge: 10000 34
Centrifuge: 17000 34
?. [Genomic DNA isolation]
Discard the flow-through and centrifuge again at maximum speed for .
?. [Genomic DNA isolation]
Place the spin column into a 1.5 ml tube and add  nuclease-free water to the center of the column. Spin at  for .
100 µl
Centrifuge: 9000 34
Centrifuging at 9000 x g reduces the chance of breaking off the Eppendorf tube’s lid.
?. [Genomic DNA isolation]
Quantify DNA using NanoDrop and store at .
-20 °C
?. [Genomic DNA PCR]
Make a PCR master mix by adding all the components except genomic DNA for the required number of reactions:PCR reaction mix for one sample:  AB1Genomic DNA (10ng/µl) 5.0 µl210 µM Primer F 1.0 µl310 µM Primer R or R 2 1.0 µl410X High Fidelity buffer 2.5 µl5DMSO 0.5 µl610 mM DNTP 1.0 µl750 mM MgSO4 1.0 µl8Platinum Taq DNA Polymerase High Fidelity 0.5 µl9Water 12.5 µl
A wide variety of Taq DNA polymerase products are available for PCR. Each may need a slightly different PCR condition. We recommend Platinum Taq DNA Polymerase High Fidelity for validating ARF-TIR1 genomic DNA insertion using PCR. The forward and reverse primers flank the genomic DNA integration site of the ARF-TIR1 construct (Figure 2A). If there is no insert, the PCR produces a smaller product with the flanking primer, but yields a larger product if ARF-TIR1 is properly integrated (details below). Heterozygous integration of the construct results in the two bands. Independently, we also perform a PCR using the flanking forward primer and a primer that is internal to the insert to confirm the integration of the construct at the locus (Figure 2B).
AB1Genomic DNA (10ng/µl) 5.0 µl210 µM Primer F 1.0 µl310 µM Primer R or R 2 1.0 µl410X High Fidelity buffer 2.5 µl5DMSO 0.5 µl610 mM DNTP 1.0 µl750 mM MgSO4 1.0 µl8Platinum Taq DNA Polymerase High Fidelity 0.5 µl9Water 12.5 µl
?. [Genomic DNA PCR]
Aliquot  of the master mix into 0.2 ml PCR tubes and add  of  genomic DNA into each reaction mix. Use the parental HEK293T DNA as a negative control.PCR condition as follows: Initial denaturation 30 cycles: Final extension  hold ∞Primers used AAVS1GenomicF (F) 5'-CTGCCGTCTCTCTCCTGAGT-3' AAVS1GenomicR (R) 5'-ACAGTTGGAGGAGAATCCACC-3' Internal Primer (R2) 5'-ATTATGATCTAGAGTCGCGGC-3'
20 µl
5 µl
95 °C
95 °C
59 °C
68 °C
68 °C
4 °C
?. [Electrophoresis]
Make  agarose gel with 1X TAE buffer.
?. [Electrophoresis]
Add  6x DNA sample buffer to the PCR tubes and load onto the 1% agarose gel.
5 µl
?. [Electrophoresis]
Run the samples at constant 90V for .
?. [Electrophoresis]
Stain the gel with SYBR Safe DNA gel stain diluted at 1:10000 with 1x TAE buffer for  and wash twice with 1x TAE for  each.
?. [Electrophoresis]
Visualize the bands using a UV transilluminator (Figure 2A&B).
The negative control produces an amplicon of 1892bp with genomic F and R primers. Successful integration of the construct homozygously produces an amplicon of 7256bp, and heterozygous integration shows both amplicons. The PCR using the AAVS1GenomicF (F) and Internal Primer (R2) primers produce a 4677 bp amplicon only in the integrated cells, not in the negative and control cells (Figure 2A&B).
?. [Confirmation of the clones by Western blotting]
Select homozygously integrated ARF/TIR1 clones from the PCR screen.
Integration of the ARF and TIR1 genes at the AAVS1 locus does not necessarily mean the genes are expressed. Use Western blotting to test whether ARF and TIR1 proteins are present in the cell.
?. [Confirmation of the clones by Western blotting]
Thaw the frozen protein lysate (Step 25) and heat again at  for  to .
95 °C
?. [Confirmation of the clones by Western blotting]
Separate proteins by loading  of the samples on a  acrylamide gel, transfer the proteins onto nitrocellulose or PVDF membrane, block membrane with  nonfat dry milk, and probe with anti-GFP or anti-HA and anti-TIR1 antibodies. β-Actin (1:5000; Sigma, A1978) can be used as a loading control. Include parental cell lysate as a negative control (Figure 2C).
10 µl
After insertion and expression are confirmed by genomic PCR and Western blot, the construct should be sequenced to confirm that mutations were not incorporated during the process. This cell line will serve as the progenitor cell line for tagging genes of interest with full-length AID.