STRAIGHT-IN Line Procedure

This protocol describes the step-by-step integration of Donor plasmids using the STRAIGHT-IN (Dual) platform to generate genetically modified hiPSC lines. The procedure begins with a STRAIGHT-IN hiPSC acceptor line, derived from either LU99 (from Leiden) or iPS11 backgrounds. We currently possess two types of acceptor lines:

  • Single landing pad lines: iPS11 background only

  • Dual landing pad lines: iPS11 at CLYBL and LU99 at either CLYBL or AAVS1

Two landing pad systems are used:

  • Bxb1-GT landing pad

    • Confers zeocin (bleomycin) resistance upon integration

    • Carries CAG-BFP (bright blue signal in iPS11 Single GT line or iPS11 Dual line) or PGK-BFP (silenced in both LU99 lines)

    • Auxiliary sequences (BFP, BleoR, plasmid backbone) are excised using Cre recombinase

  • Bxb1-GA landing pad

    • Confers puromycin resistance upon integration

    • Carries CAG-GFP (bright green signal in iPS11 Single GA line) or CAG-mScarlet (bright red signal in iPS11 Dual line) or PGK-mScarlet (silenced in both LU99 lines)

    • Auxiliary sequences (GFP/mScarlet, PuroR, plasmid backbone) are excised using Flp recombinase

Key Reagents (TC Supplies)

  • 24-well plates

  • rhLaminin-521: aliquots are in Grand Paddie (-20°C) and stocks in Sven (-20°C).

  • PBS +/+: we keep the stock in cell culture fridge and in Oaken (4°C).

  • PBS -/-

  • Gentle Cell Dissociation Reagent

  • Penicillin/Streptomycin: aliquots and stocks are in Olaf (-20°C).

  • StemFlex medium: aliquots and stocks in Sven (-20°C). It is aliquoted without Penicillin/Streptomycin but once the aliquot of the medium has been thawed, add Penicillin/Streptomycin (1:100). Do not thaw StemFlex medium at 37°C, do it at room temperature or preferably at 4°C overnight.

  • Neutralization medium: DMEM/F12 + 1% FBS

  • RevitaCell Supplement: used at 1:200. Aliquots are in Grand Paddie (-20°C) and stocks in Sven (-20°C).

  • OptiMEM: aliquots and stocks are in Oaken (4°C).

  • Lipofectamine Stem Transfection Reagent: stocks are in Oaken (4°C).

  • Zeocin™ selection reagent: aliquots are in Grand Paddie (-20°C). Stock is at 100 mg/mL and the working solution is 15 µg/mL (1:6666).

  • Puromycin: aliquots are in Grand Paddie (-20°C). Stock is at 1 mg/mL and the working solution is 1 μg/mL (1:1000).

  • DNeasy Blood and Tissue kit

  • STRAIGHT-IN acceptor lines: LU99 CLYBL Dual, LU99 AAVS1 Dual, iPS11 CLYBL Single GT, iPS11 CLYBL Single GA and iPS11 CLYBL Dual

Key Reagents (Plasmids and modRNA)

  • eeBxb1 plasmid (pKG3466) or eeBxb1 modRNA (pKG3471). Miniprep fresh plasmid or preferably use modRNA. Frozen aliquots of the modRNA should be stored at -80°C (Elsa) in the box with the rest of the recombinases.

  • p53DD modRNA (pKG3199). Frozen aliquots of the modRNA should be stored at -80°C (Elsa) in the box with the recombinases.

  • Flp-T2A-BleoR modRNA (pKG3212). Frozen aliquots of the modRNA should be stored at -80°C (Elsa) in the box with the recombinases.

  • TAT-Cre protein: aliquots can be found at -80°C (Elsa) in the box with the recombinases.

  • Cre modRNA (pKG1779, pKG3213, pKG3214 or pKG3717). Frozen aliquots of the modRNA should be stored at -80°C (Elsa) in the box with the recombinases.

  • Donor plasmids containing attB-GT or attB-GA and the desired DNA cargo

General Preparation

Before starting any step, pre-warm all culture media for 15 to 30 minutes at room temperature.

Pre-warm:

  • StemFlex (with or without antibiotics)

  • Neutralization medium

  • OptiMEM

STRAIGHT-IN Integration Protocol

Two Days Before Transfection (d -2)

  1. Dilute rhLaminin-521 at 1:20 in PBS +/+.

  2. Add 300 µl per well to a 24-well plate (15 µl rhLaminin-521 + 285 µl PBS +/+).

  3. Seal with Parafilm and incubate overnight at 4 C.

  • Alternatively, coat on the day of splitting and incubate for 2 hours at 37°C.

Day Before Transfection (d -1)

  1. Warm the coated plate at 37 C for 10 minutes.

  2. Select a hiPSC well that is approximately 80% confluent.

  3. Aspirate StemFlex.

  4. Rinse once with 1 mL PBS -/-.

  5. Rinse again with 1 mL PBS -/-.

  6. Add 300 to 500 µl of Gentle Cell Dissociation Reagent.

  7. Incubate 5 minutes at 37°C with 5% CO2.

  8. Aspirate the dissociation reagent and add 1 mL StemFlex + RevitaCell (1:200).

  9. Gently triturate 5 to 10 times to obtain a single cell suspension and transfer to a tube containing 1.5 to 2 mL neutralization medium.

  10. Centrifuge at 300g for 3 minutes.

  11. Aspirate supernatant and resuspend in 0.5 mL StemFlex.

  12. Count cells and seed 100K cells per well (24-well scale) on rhLaminin-521 coated wells. * This number works well for our lines, but can be optimized between 150K-300K.

  13. Plate cells and distribute evenly by gentle rocking. Incubate at 37°C and 5% CO2.

Day of Transfection (d0)

  1. Confirm cells are approximately 30 to 40% confluent.

  • If too confluent, re-seed.

  • If too sparse, feed and wait until they reach the appropriate density.

  1. Prepare transfection mixes. Per well:

  • 600 ng Donor plasmid

  • 400 ng eeBxb1 modRNA

  • 400 ng p53DD modRNA

  • 3 µl Lipofectamine Stem reagent

  • Prepare Lipofectamine master mix based on n + 0.5 wells.

  • Add DNA mix to Lipofectamine mix (not the reverse).

  1. Incubate 50 µl Lipofectamine:DNA mix for 10 minutes at room temperature.

  2. Aspirate StemFlex, rinse once with PBS -/- and add 0.5 mL OptiMEM.

  3. Add 50 µl transfection mix dropwise and gently shake.

  4. After 4 hours, add 0.5 mL StemFlex without removing OptiMEM.

Lipofectamine Stem Transfection Overview (24-well format)

Step

Tube

Components

Amount per Well

1

Tube 1

OptiMEMLipofectamine Stem Reagent

22 µL3 µL

2

Tube 2

OptiMEMDNA

25 µL - DNA volume600 ng donor + 400 ng Bxb1 + 400 ng p53DD

3

Add Tube 2 into Tube 1 and mix well

4

Incubate 10 minutes at room temperature

5

Rinse cells with PBS -/- and add 0.5 mL OptiMEM

6

Add 50 µL complex to each well and swirl to distribute

7

Incubate 4 hours at 37°C with 5% CO₂

8

Add 0.5 mL warm StemFlex per well and incubate overnight

One Day After Transfection (d1)

  1. Aspirate media, rinse once with PBS -/-, add 0.5 mL StemFlex.

  2. Expect some cell death due to plasmid toxicity.

  3. Assess fluorescence if applicable.

Two Days After Transfection (d2)

  1. Begin antibiotic selection if cells are healthy and above 50% confluent.

  • Zeocin 1:6666 for Bxb1 GT

  • Puromycin 1:1000 for Bxb1 GA

  1. Prepare at least 1.5 mL antibiotic medium for multiple refreshments. Keep at 4°C for up to one week.

Three to Four Days After Transfection (d3 to d4)

  1. Rinse once with PBS -/- and add 0.5 mL antibiotic medium.

Note

A lot of cell death during selection is expected.

Five Days After Transfection (d5)

  1. Look for clear colonies. If present, stop antibiotic selection.

Note

  • Three days of selection are usually sufficient.

  • Include a negative control lacking eeBxb1 and p53DD to confirm full lethality.

  1. Replace with 0.5 mL StemFlex without antibiotic.

Six to Eight Days After Transfection (d6 to d8)

  1. Refresh medium every other day.

  2. Proceed to the STRAIGHT-IN Excision Protocol.

STRAIGHT-IN Excision Protocol

The excision strategy depends on the landing pad:

  • GA landing pad: excised using Flp recombinase

  • GT landing pad: excised using Cre recombinase

Each system has two workflow options: A (no replating) and B (replate cells).

Flp-Mediated Excision (GA Landing Pad)

Option A: Direct Transfection (d6 to d8)

  1. Once colonies are large and healthy, transfect with Flp-T2A-BleoR modRNA.

  2. Per well, use:

  • 500 ng Flp-T2A-BleoR modRNA

  • 2 µl Lipofectamine Stem

  1. Prepare Lipofectamine master mix based on n + 0.5 wells. Add RNA mix to Lipofectamine.

  2. Incubate 10 minutes at room temperature.

  3. Aspirate medium, rinse with PBS -/-, add 0.5 mL OptiMEM.

  4. Add 50 µl Lipofectamine:RNA mix.

  5. After 4 hours, add 0.5 mL StemFlex without removing OptiMEM.

  6. After another ~2-4 hours, add 0.5 mL StemFlex containing Zeocin (1:6666).

  • Selection may also begin the next day if needed.

Option B: Replate and Transfect (d6 to d8)

  1. Replate cells into two rhLaminin-521 coated wells, splitting evenly.

  2. When wells reach 30 to 40 % confluency, proceed.

  3. Transfect only one well with Flp-T2A-BleoR modRNA. Keep the other as a contingency.

  4. Follow steps from Option A, starting at step 2.

Cre-Mediated Excision (GT Landing Pad)

Option A: Replate and Use TAT-Cre Protein (d6 to d8)

  1. Replate cells into two rhLaminin-521 coated wells.

  2. When cells reach 30 to 50% confluency, proceed.

  3. Prepare a 0.5 mL StemFlex mixture containing 2.25 µl TAT-Cre (1 µM final).

  • Mix the components in a separate protein low-bind/retention tube before adding to cells.

  1. Add the mix to one well. Keep the second well as a backup.

  2. The next day, rinse with PBS -/- and replace with 0.5 mL StemFlex.

  3. Allow cells to grow until confluent, typically 2 days. Refresh every other day.

  4. Passage cells and collect samples for gDNA extraction and CNV ddPCR assays for BleoR and EBFP2/EGFP.

  • Typical excision efficiency is 50 to 60%.

  • Additional rounds of TAT-Cre may further increase excision rates.

Option B: Replate and Use Cre modRNA (d6 to d8)

  1. Replate cells into two rhLaminin-521 coated wells, splitting evenly.

  2. When wells reach 30 to 40% confluency, proceed.

  3. Transfect only one well with Cre modRNA. Keep the other as a contingency.

  4. Follow steps from Option A, starting at step 2.

  5. The next day, rinse with PBS -/- and replace with 0.5 mL StemFlex.

  6. Allow cells to grow until confluent, typically 2-3 days. Refresh every other day.

  7. Passage cells and collect samples for gDNA extraction and CNV ddPCR assays for BleoR and EBFP2/EGFP.

  • Typical excision efficiency is 30%.

  • Additional rounds of Cre modRNA transfection may further increase excision rates.