Evolution of our Cloning Strategy

Initially wanted to follow GAPTrap paper to the T

Use GAPTrap – mCherry- IRES- Hygro as backbone

• Ampicillin and hygromycin resistance

Clone in sACE 2 from twist plasmid using AscI and SalI digestion

• ACE 2 plasmid has ampicillin resistance
• ACE 2 replaces mCherry

Form GAPTrap guide RNA and Cas 9 mRNA

Electroporate into iPSC

Changed to Geng et al.’s CRISPR method and considered lipofectamine

Form guide RNA dimer

Clone into a Addgene’s PX459V2.0-eSpCas9 plasmid (#108292) by BbsI digestion

• Much faster CRISPR method since using single plasmid
• Also selects cells by high dose puromycin = faster selection

Lipofectamine transfection

• Plasmids/DNA will be coated in a lipid layer forming a liposome, which then can merge into cell membranes without use of electric current
• Cheaper, easier

Met with Ed Stanley July 24th

Suggested using GAPTrap- mtagtdTom-IRES-Muro

• tdTomato fluoresces much brighter than mCherry

• Uses puromycin, which selects better than hygromycin

Change antibiotic resistance of ACE 2 plasmid to kanamycin/chloramphenicol

• Different resistance to the GAPTrap plasmid’s ampicillin resistance = more efficient selection

Check Restriction enzyme cut sites

• Suggested phosphatase treatment to make sure ligation is directional in case enzymes don’t work well together

Will provide us with TALENs plasmids

• No need for making a CRISPR plasmid
• GAPDH has a lot of pseudogenes so TALENs’ specificity makes it more ideal than CRISPR

Suggested using electroporation instead of lipofectamine

• Lipofectamine = aggregating reagent, risk of multiple inserts since no guarantee that each liposome doesn’t contain other fragments/junk
• Suggested doing a trial electroporation run with uncloned plasmids to check that we’re not too dumb and can select for some cells

Request for more bacteria when ordering plasmids so we don’t have to grow them up ourselves

Should we transfect macrophages or transfect iPSCs then differentiate them into macrophages afterwards?

• Macrophages are designed to reject foreign DNA so better to transfect iPSCs

Use Nadia’s protocol for differentiating iPSCs to macrophages after they’ve been transfected and verified

But Gibson assembly requires fragments of >200 nucleotides

• HA tag is 27 bp
• P2A (57 bp) and T2A tags (54bp)

So if we did use Gibson, would need to order constructs of T2A-ACE2-tag and P2A-tdTom

• And gibson involves additional step of PCR to verify primers

Decided instead to order them as a single synthesised construct using the sponsorship from Twist

Later on, we can use Gibson to rearrange order of cistrons and see how that affects expression levels