Weiss Lab:Notebook/Non-Integrating Lentiviral Vectors

Site-directed Mutagenesis
November, __, 2009 Used QuickChange Multi Site Directed Mutagenesis Kit from Strategene

Three Mutants: 1. IN D64V 2. IN D64V, N120L, Q148A, and W235E 3. IN K264, K266R, K273R

Primers:

Mini-prep
1. Culture isolated colonies from plates in liquid broth over night.

DNA Sequencing
November 23, 2009

1. Dilute primer to 100 uM.
 * oS-LW-INT-RV @ 30.8nmole. Add 308 uL to make 100 uM stock. Dilute to 8 uM (add 1uL of stock to 11.5 uL of sterile water).
 * oS-LW0INT-FW @ 27.5nmole. Add 275 uL to make 100 uM stock. Dilute to 8 uM (add 1 uL of stock to 11.5 uL of sterile water).

2. Add 11 uL of DNA with 1 uL of primer at 8 uM DNA concentrations:
 * Mut 2-1 @ 102.3 ng/uL --> 1125 ng
 * Mut 2-2 @ 40.9 ng/uL --> 449.9 ng
 * Mut 2-3 @ 87.9 ng/uL --> 966.9 ng
 * Mut 3-1 @ 52.6 ng/uL --> 578.6 ng
 * Mut 3-2 @ 241.5 ng/uL (Digest of this did not produce correct fragments)
 * Mut 3-3 @ 104.2 ng/uL --> 1146 ng

3. Send to sequencing center before 4pm! Orders: LW 01: Mut2-1 + FW primer LW 02: Mut2-1 + RV primer etc...

DNA Sequencing Data Analysis
November 26, 2009 (Thanksgiving!)

Results from sequencing:
 * Using reverse primer (oS-LW-INT-RV) gave no sequencing results.
 * Fwd primer (os-LW-INT-FW) gave good alignment results, but sequencing data stopped abruptly after the Integrase gene which is at the end of ORF3. There was no gradual loss of resolution of bases and etc.
 * This leads us to believe that the segment immediately after ORF3 might not exist in this plasmid. This segment is where the reverse primer should have annealed to.

For Mutant 2's, wanted D64V, N120L, Q148A, and W235E. Mut 2-1 had W235E Mut 2-2 had D64V, N120L, and W235E Mut 2-3 had D64V, and W235G

For Mutant 3's, wanted K264, 266, 273R. Mut 3-1 had K264, 266R Mut 3-2 had K266R Mut 3-3 had no mutations.

Transformation of Mut 2-2, 3-1, d1-EGFP, d2-EGFP to amplify plasmids.

 * Add 1 uL of plasmid DNA to 20 uL of XL-10 competent cells.
 * Incubate on ice for 40 min.
 * Add 200 uL of SOC media.
 * Plate 20 uL of cells on AmpR agar plates and incubate at 37 C for > 16 hours.

Isolate more colonies from 1st mutagenesis reaction

 * Make 5 culture tubes with 5 mL agar and 1:1000 Amp.
 * Add colonies respectively: Mut 2-4, 2-5, 2-6, Mut 3-4, 3-5
 * Incubate at 37C for > 16 hours.

November 28, 2009

 * Mini-prep 2-4, 2-5, 2-6, 3-4, 3-5. Used 3mL of cell culture, spun down at 13kRPM for 2 min.
 * Observed isolated single colonies for Mut 2-2, 3-1, d1-EGFP, d2-EGFP! =) Will midi-prep on Monday. Put at 4C.

November 30, 2009
Midi-prep of d1-EGFP, d2-EGFP, Mut 2-2 and 3-1.
 * Mut 2-2: 356.3 ng/uL
 * Mut 3-1: 327 ng/uL

December 5, 2009
Virus Packaging:
 * 148 (WT pol gene): pCMV-dR8.2-dvpr
 * Need 2.8 ug from 750 ng/uL --> 3.73 uL
 * 149 : pCMV-VSV-G
 * Need 1.4 ug from 1186 ng/uL --> 1.18 uL
 * EGFP
 * Need 1.1 ug from 302 ng/uL --> 3.64 uL

Wednesday, December 9 and Saturday, December 12
FACS analysis of infected 293FT cells


 * Prepare a 12-well dish with 1 mL of media
 * Add 200 uL trypsin per well of infected plate, incubate 3 min.
 * Rescue with 1mL media, pipetting up and down well. Add 1000 uL (a little less) to FACS tube, and add remaining cells to the new plate.
 * Keep cells on ice.


 * FACS settings: event count for first day: 2000 (time constraint after solving a lot of machine issues). Event count for second day: 15000.

Wednesday FACS data: ... etc. Did not save negative control or Mut2-2n1 data.
 * .001 file : Mut2-2n2

Tuesday, December 15
Event count is different for all samples (usually around 10000) because it's too slow. Order:
 * Neg control
 * Mut 2-2 n1
 * Mut 2-2 n2
 * Mut 2-2 n3
 * Mut 3-1 n1
 * Mut 3-2 n2 (**lowest: only 5000 events)
 * Cntrl n1 (event count: 8100)
 * Cntrl n2 (event count: 5000)

Observations:
 * Mut 2-2 show a narrower distribution of GFP (between 10^1 and 10^2 of FL1-H, which is the GFP channel. Nice peak curve on the histogram). GFP expression is definitely lower than Mut 3-1 and the Control Integrase package.
 * Mut 3-1 show a wide distribution between 10^1 and 10^4. GFP expression is lower, but probably not as low as Mut 2-2.
 * Control: Distribution between 10^1 and 10^4. Probably multiple viruses affecting one cell. GFP expression is still strong. Low cell count though.