Expanding the genetic code of a mouse Mito

Expanding the genetic code of a mouse Mito Lab Soonjag Lee

Why we do Expansion of genetic code ? PTM (Post translational modification) 1. provide tools to research the function of proteins 2. improve the activity of protein drugs

To make acetylated lysine into protein Ac. K

Normal translation Process GFP TAG

Translation of expandig genetic code Ac. K Acetyl lysine Ac. KRS Acetyllysine t. RNA synthetase t. RNA

Translation of expandig genetic code Ac. K GFP TAG

Transgenic Mouse Constructs Ac. KRS GFP

Strategy for making transgenic mouse Ac. KRS GFP

Ac. KRS Making transgenic mice

14 15 1 16 2 17 3 18 4 19 20 5 21 6 22 7 23 24 8 25 26 9 10 27 28 11 12 Co n tro l Establish transgenic lines : Ac. KRS _ Founder 29 30 31 Ack. RS Geno 2 32 33 13

nt ro l Establish transgenic lines : GFP _ Founder 2 ’ 3 ’ 4 ’ 5 ’ 6 ’ 7 ’ 8 ’ Co nt ro l Co 1 ’ 3 4 5 6 7 8 9 10 11 12 13 14 nt ro l 2 Co po si tiv e 1 27 28 29 30 31 32 33 34 35 36 37 15 16 17 18

Strategy for making transgenic mouse Ac. KRS GFP Ac. KRS. GFP mouse

Conformation of Transgenic mouse

MEF(Mouse Embryonic Fibroblasts) from Ac. KRS. GFP mouse

MEF(Mouse Embryonic Fibroblasts) from Ac. KRS. GFP mouse No detectable GFP induced by Ac. K Optic image MEF (Ac. KRS, GFT 39 T) (-) Ac. K ( + ) Ac. K

Translation of expandig genetic code Ac. K GFP TAG

Why GFP is not detected in MEF(Ack. RS. GFP)? 1. Ac. K internalization? 2. Degradation of Ac. K? 3. NMD(Nonsense-mediated m. RNA decay)? Nonsense-mediated m. RNA decay (NMD) is a translationcoupled mechanism that eliminates m. RNAs containing premature translation-termination codons

UPF 2 is a factor of NMD(Nonsense-mediated m. RNA decay)

1. Ac. K internalization? MEF(Ac. KRS. GFP) - Ac. K vs. CF 3 -Ac. K (+) CF 3 -Ac. K

2. Degradation of Ac. K? NAM( Nicotinamide : Deacetylase inhibitor)-treated (+) Ac. K / NAM

Conformation of UPF 2 Knockdown in MEF(Ac. KRS. GFP)

3. Nonsense-mediated m. RNA decay? - UPF 2 Knock down (+) Ac. K UPF 2 KD

FACS analysis - (+) Ac. K vs (+) Ac. K / UPF 2 KD Ctrl UPF KD Fluorescence GFP-A Geo Mean(P 2) Relative fluorescence (%) Cell counts Ctrl 160 *** 140 120 100 80 60 40 20 0 control KD(UPF 2) UPF KD

Confocal microsope (-) Ac. K (+)Ac. K UPF 2 KD

MEF(Ac. KRS. GFP), organ cryosection / slide scanner

Thank you