mouse iPSCs with lentivirus

Culturing

Thaw 293TN cells

02/07/2013

Do not reach confluency. Myco test

Split 293TN cells

02/08/2013 - 02/11/2013

When cells reach 70-80% confluency split at least 1/3 or 1/4

Seed 3.5 million 293TN cells per P60

02/12/2013

at least one P60 per plasmid. Use MEF media without antibiotics

Morning: Seed fibroblast to P60

02/13/2013

500,000 cells per P60

Split onto 100mm MEFs

02/19/2013

One well to two P100.

Change to mouse+DOX media

02/21/2013

Check for green iPSC colonies

02/21/2013 - 03/04/2013

Isolate iPSc colonies transfer to 12 well MEF

02/21/2013 - 03/04/2013

expand clones

03/04/2013 - 03/08/2013

Split good clones and take off DOX

03/07/2013

Freeze vials of good iPSC clones

03/07/2013

Freeze/expand clones that grow well w/o DOX

03/11/2013

Transfection

Transfect 293TN with plasmids

02/13/2013

No media change prior

24 hr Psuedo-viral media collection

02/14/2013

Collect and filter media from 293 TN cells

12hr after transfect: Change with antibiotic free media

02/14/2013

12 hours after transfection

48 hr psuedo viral media collection

02/15/2013

Filter

72hr psuedoviral media collection

02/16/2013

transduction

24hr Transduction of Fibroblast

02/14/2013

Add Fitered psuedo-viral media+polybrene to fibros

48hr Transduction of Fibroblast

02/15/2013

72hr Transduction of Fobroblasts

02/16/2013

Change to antibiotic free MEF media

02/17/2013

No viral particles, no antibiotics