The document discusses cloning issues and solutions for constructing a lentiviral vector. It describes problems with restriction sites that prevented adding a second insulator. Sequencing between the polyadenylation sites found no sequence. A patch was designed with new restriction sites. Testing found the eGFP-Neo cassette was non-functional, so it will be replaced with eGFP-Zeo. The final construct design is shown. Further IHC approaches for detecting CRE are discussed.
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2011 4.14
1. hPGK
1
eGFP-Neo
2
6 8 3 PolyA
Ins
PolyA
mCherry
Jay Gantz, Laflamme Lab Meeting
April 14, 2011
2. What I told you on March 2nd...
EF1a-CRE lenti plasmid is done, need to make
virus, titer and test
CK7-CRE seemed to work but IHC needed
Cloning: problems with restriction sites (dam
methylation), several lucky breaks, one last step
2
3. Let’s start with the cloning
7 4 5+6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Thought there was one last step: 2nd insulator
3
4. Let’s start with the cloning
7 4 5+6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Thought there was one last step: 2nd insulator
However: cut site between pAs doesn’t cut. Uh oh.
3
5. Let’s start with the cloning
7 4 5+6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Thought there was one last step: 2nd insulator
However: cut site between pAs doesn’t cut. Uh oh.
No other sites. No obvious options.
3
6. Let’s start with the cloning
7 4 5+6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Thought there was one last step: 2nd insulator
However: cut site between pAs doesn’t cut. Uh oh.
No other sites. No obvious options.
Tried sequencing between PolyAs: nada
3
7. Solution:
7 4 5+6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
4
8. Solution: A Patch!
7 4 5+6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
4
10. Solution: A Patch!
7 4 5+6 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Patch
Patch advantages: •Perfect sequence (we ordered the
oligo)
•Engineered multiple new and
unique restriction sites around all
components
4
11. Solution: A Patch!
7 4 5+6 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Patch
Patch advantages: •Perfect sequence (we ordered the
oligo)
•Engineered multiple new and
unique restriction sites around all
components
Patch disadvantage: Time (ordered mid-March, any
day now...)
4
12. Meanwhile... tested to look for GFP
7 4 5+6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Idea: check if right half is functional in HEKs
5
13. Meanwhile... tested to look for GFP
7 4 5+6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Idea: check if right half is functional in HEKs
Transfected (along with a + control)
5
14. Meanwhile... tested to look for GFP
7 4 5+6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Idea: check if right half is functional in HEKs
Transfected (along with a + control)
No green.
5
15. Why no green?
7 4 5+6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Reason 1: low quality DNA prep (P/C made a huge
difference for Nathan)
6
16. Why no green?
7 4 5+6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Reason 1: low quality DNA prep (P/C made a huge
difference for Nathan)
Reason 2: mutation? but we have ok sequence
6
17. Why no green?
7 4 5+6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Reason 1: low quality DNA prep (P/C made a huge
difference for Nathan)
Reason 2: mutation? but we have ok sequence
Reason 3: look into the construct further...
6
18. eGFP-Neo
Advertised as a fusion protein.
Looking closer at the sequence:
This is not a fusion protein.
Called OligoEngine...
7
20. Solution: eGFP-Zeo!
PLoS One. 2009; 4(8): e6529.
A Versatile Viral System for Expression and
Depletion of Proteins in Mammalian Cells
Campeau et al.
A true fusion protein!
8
34. Source of EF1a-CRE problem?
A: dirty DNA leading to empty or non-functional viruses
B: Bad sequence or backbone
I did a P/C got clean DNA and submitted for full
sequencing
21