Rogi1 and Rogi2 LPs

We currently have monoclones for v2 and v3. The table below highlights the key attributes of each LP architecture:

Line summary

V2 Rogi1/Rogi2 dual LP RMCE line:

locus	pHA for cargo	recombinase	positive selection (gene, drug)	counterselection (gene, drug)
Rogi1 LP	pKG01862	Cre	N/A	iCasp9, AP1903
Rogi2 LP	pKG02278	Bxb1	N/A	HSV-TK SR39h, GCV or PCV

V3 Rogi1/Rogi2 dual LP single site integrase line:

locus	pHA for cargo	recombinase	positive selection (gene, drug)	counterselection (gene, drug)
Rogi1 LP	pKG02180	Bxb1	BsdR, Blasticidin	HSV-TK SR39h, GCV or PCV
Rogi2 LP	pKG02181	PhiC31	BleoR, Zeocin	iCasp9, AP1903

Integration into landing pad lines

Note

These protocols are still being optimized! Important variables that need optimization include: 1) the ratio of recombinase to donor plasmid 2) dosing schedule of drug selection/counterselection 3) scale needed to obtain a sufficient number of recombined cells

Day -1

Seed cells at 120k per 24 well.

Day 0

Transfect the donor DNA, using Mirus transfection reagent.

Note

The full Mirus manual is here if you have questions.

1. Make a condition mix by combining 500 ng of plasmid donor DNA (per 24 well) with 50 µL (per 24 well) of OptiMEM reduced serum media, mixing gently via pipetting.

Important

Use OptiMEM, not knockout DMEM! It really does give better results.

2. Add 3 µL of Mirus TransIT-LT1 reagent per µg of donor DNA. That is, add 1.5 µL of transfection reagent per 24 well to the condition mix. Pipette gently to mix.

3. Let the condition mixes incubate at room temperature for 15 to 30 minutes.

4. Add 50 µL of each condition mix to each 24 well, dropwise. Gently rock the media back and forth.

Day 1

Transfect the modRNA. You do not need to media change after the DNA transfection.

Note

The full MessengerMAX manual is here if you have questions.

1. Determine how much recombinase mass you need to add:

   Recombinase	Mass per 24 well	Recombiase:donor plasmid ratio
   Bxb1	100 ng	1:4
   PhiC31	100 ng	1:4
   Cre	6.25 ng	1:64

2. Create Solution 1 condition mixes by combining recombinase modRNA with 20 µL of OptiMEM per 24 well.

3. Create Solution 2 condition mixes by combining 2 µL of MessengerMAX per µg of modRNA added in the previous step with 20 µL of OptiMEM. Incubate at room temperature for ten minutes.

4. Add solution 1 to solution 2 for each condition mix, and incubate for 5 minutes.

5. Add 40 uL of the resulting condition mix dropwise to the wells.

Day 2

Media change the cells to fresh DMEM.

Day 5

Begin selection. The recommended concentrations for selection are:

Selection	Quantity
Puro	10,000x
Zeocin	100x

The recommended concentrations for counterselection are

Counter-selection	Concentration	Dilution
GCV	10 µM	1000x
AP1903	10 nM	1000x

Day n

Continue selecting / passaging until you get a sortable population.