CENTER FOR REGENERATIVE MEDICINE of BOSTON UNIVERSITY and BOSTON MEDICAL CENTER
670 Albany Street
nd
2 Floor
Boston, MA 02118
Tel: 617-414-2969
http://www.bumc.bu.edu/stemcells/
iPSC CORE
Excision of STEMCCA vector from hiPSCs by transient expression of Cre-recombinase
In this protocol, hiPSC clones generated with STEMCCA-LoxP lentivirus are transfected with
a plasmid vector expressing Cre-recombinase and the Puro resistance gene. Following a twoday selection with Puromycin, hiPSC colonies are picked, expanded and screened for the
presence of the lentiviral cassette.
Preparation of hiPSCs for transfection
hiPSCs are grown in 6-well plates containing inactivated Puromycin-resistant MEFs and hESC
medium. The density and size of the hiPSC colonies at the time of transfection are the most
critical factors influencing the efficiency of excision. Thus, when preparing cells for Cre
excision, plate cells at different dilutions in a 6-well plate (e.g. 1:20,1:40,1:60) and culture for 5
days. Choose a well containing 30-50 mid-sized colonies.
Note: If Puromycin-resistant feeder cells are not available, regular MEFs can be used.
Although some feeders are eliminated during the 2-day Puromycin treatment, more MEFs can
be added later if necessary.
You will need:
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TransIT-HeLaMONSTER™ Transfection Kit (Mirus Cat# MIR2900)
DMEM/F12 (Life Technologies Cat# 11330-032)
Plasmid pHAGE2-EF1alpha-Cre-IRES-Puromycin-W
hESC medium
Puromycin
Day 1: Transfection
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In a 1.5 ml tube add 200 ul of DMEM/F12 and 6 ul of TransIT HeLa Reagent. Mix and
incubate the tube at RT for 10 minutes.
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Add 2 ug of the plasmid pHAGE2-EF1a-Cre-IRES-Puro-W. Mix and incubate the tube
at RT for 10 minutes.
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Add 3 ul of Monster Reagent. Mix and incubate the tube at RT for 10 minutes.
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Add the mixture to the cells, containing 2 ml of fresh hESC medium, and incubate in a
37 °C, 5% CO2 incubator ON.
670 Albany Street, 2nd Floor. Boston, MA 02118. 617-414-2969.
Day 2: Remove hESC medium and add fresh medium containing 1ug/ml of Puromycin.
Day 3: Remove hESC medium and add fresh medium containing 1ug/ml of Puromycin.
Day 4: Remove medium and add hESC medium WITHOUT Puromycin.
Puromycin treatment is performed for 2 days ONLY! Transient expression of Cre
recombinase is sufficient to excise the STEMCCA vector.
Day 5 to day 14: Change medium (without Puromycin) daily. Although most cells are
sensitive to puromycin, some of them in the periphery of the colonies are effectively
transfected and survive the two-day puromycin selection. These will start forming new
colonies that can be picked around day 14 for expansion and analysis (see picture below).
Day 15: Manually pick 6 colonies in a 12-well plate containing feeders (regular MEFs). After
around a week, split cells at ~1:5-1:10 into a new 12-well feeder plate and use the remaining
cells for gDNA extraction and PCR screening.
Generation of transgene-free iPSCs. iPSCs are transfected with a plasmid coexpressing
Cre recombinase and a Puromycin resistance gene, as described in Somers et al, 2010. A)
iPSCs before transfection. B) Cell death is observed after two days of Puromycin selection C)
New colonies emerge from resistant cells and are ready to pick around 10-14 days after
transfection.
670 Albany Street, 2nd Floor. Boston, MA 02118. 617-414-2969.
PCR screening
Primers:
pcr exc 5’: 5’-TGG CTC TCC TCA AGC GTA TT-3’ (binds to IRES)
pcr exc 3’: 5’-GTT GTG CAT CTT GGG GTT CT-3’ (binds to hSox2)
PCR reaction:
Cycling:
10x PCR buffer
50 mM MgCl2
10 mM dNTP’s
10 uM primer 5’
10 uM primer 3’
template (50 ng/ul)
Taq
H2O
Total
1
2
3
2.5 ul
0.75 ul
0.5 ul
1.25 ul
1.25 ul
2 ul
0.25
16.5 ul
25 ul
94C
94C
55C
72C
72C
3 min.
45 sec.
30 sec.
1 min.
10 min.
30 X
4
Top gel: Excision screening (expected band:
400 bp)
Bottom gel: GAPDH (expected band: 678 bp)
1: hiPSC clone before Cre excision
2, 3, 4: Three subclones after Cre excision.
670 Albany Street, 2nd Floor. Boston, MA 02118. 617-414-2969.