Organon TSSK 051104
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Transcript of Organon TSSK 051104
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Male contraceptiveMale contraceptive
Maturation
in
Epididymis
Mitosis Meiosis ???
Stem cells
Reversible??
Segregation
chromosomes
Aneuploidy??
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TSSK mouse literatureTSSK mouse literature
Tssk-1 : isolated by virtue of homology kinase domain
chr. 16
Testis-specific serine/threonine kinase
Tssk-2 identified via similar approach chr 16 tandem
Late spermatids residual bodies
Via 2-hybrid p65 substrate.
p65 is co-expressed with Tssk-1 and Tssk-2 Tssk-3 Leydig cells
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Tssk human literatureTssk human literature
Tssk-2 found on chr. 22
Tssk-1 on chr. 22 is pseudogene
Location in region linked to DiGeorge syndrome p65 homolog found in human: testis-specific (528 aa)
Tssk-3 also identified.
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Tssk-2 as target male-Tssk-2 as target male-
contraceptioncontraception Tssk-2 expressed in spermatids
Tssk-2 is testis-specifically expressed in both mouseand human
Kinase is drugable family
No Tssk-1 exists.
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Organon workflowOrganon workflow Knockout generation for functional validation:
Tssk-1/Tssk-2 double KO at Lexicon Genetics
Single KO strategy failed
Recombinant human Tssk-2 protein production:
Many attempts: E. coli, Baculo,
Recombinant human p65:
GST-fusion in E.coli
HTS development
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Tssk ISHTssk ISH
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Tssk-protein localizationTssk-protein localization
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Recombinant Tssk2
phosphorylates p65
10
1100
10
1100
10
1100
Tssk2
GST-P65 - GST
GST-P65Tssk2
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Depletion of Tssk activityDepletion of Tssk activity
Tssk2 depletion by tssk2 antibodi
0
20
40
60
80
100
120
inp inp
verdund
prot G
dep1
IgG1
dep1
tssk2
dep1
prot G
dep2
IgG1
dep2
tssk2
dep2
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P65 mutantsP65 mutants
1-216
1-88
1-164
78-216
136-216
216-592
1-592
GST-fusion p65 proteins for phosphorylation site analysis
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GST-p65 mutantsGST-p65 mutants
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HTS assayHTS assay
TSSK2 Activity
0
50
100
150
200250
300
10 100 1000 10000 100000dilution
mP
TSSK2, Ni-NTA purified
TSSK2, SEC purified
Purification of mock transfected Sf21 cells
TSSK2 DM purification
TSSK2, Ni-NTA purified, concentrated, -ATP
TSSK2, SEC purified, -ATP
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TSSK1-TSSK2 KOTSSK1-TSSK2 KO
constructionconstruction1 kb
Screening strategy:
5 external probe : NheI 3 external probe: NheI
wt: 5.2 Kb wt: 7.6Kb
targeted: 2.3 Kb targeted: 9.6Kb
Targeted allele
Neo
Frt Lox PFrtLox P
NheI NheI
5 probe 3 probe
NheI
TSSK1 TSSK2
NheI
NheI
NheI
wt allele
1 kb
Screening strategy:
5 external probe : NheI 3 external probe: NheI
wt: 5.2 Kb wt: 7.6Kb
targeted: 2.3 Kb targeted: 9.6Kb
Screening strategy:
5 external probe : NheI 3 external probe: NheI
wt: 5.2 Kb wt: 7.6Kb
targeted: 2.3 Kb targeted: 9.6Kb
Targeted allele
Neo
Frt Lox PFrtLox P
NheI NheI
5 probe 3 probe
NheI
TSSK1 TSSK2
NheINheI
NheINheI
NheINheI
wt allele
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TSSK1/2 KOTSSK1/2 KO
WT TSSK1/2 KO
Testis
Epididymis
MALE INFERTILITY
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Tssk-1?Tssk-1?
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Tssk-1 human expressionTssk-1 human expression
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Tssk-1Tssk-1
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TSSK genomeTSSK genome
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Tssk-4Tssk-4
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Human Tssk-4Human Tssk-4
TSSK-4
25x
30x
600bp-
600bp-
M Te
Ut
Ki
Th
Li
Br
He
Lu
Sp
Ov
Pa
Be
Ag
Mq
G
TSSK-4
25x
30x
600bp-
600bp-
M Te
Ut
Ki
Th
Li
Br
He
Lu
Sp
Ov
Pa
Be
Ag
Mq
G
+RT
-RT
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Mouse TSSK-4Mouse TSSK-4M A
gBr
Ep
He
Ki
Li
Mu
Te
1
Te
2
Te
3
Te
4
Lu
Sp
Pr
TSSK-4
25x600bp-
600bp-
Mq
G
30x
M Ag
Br
Ep
He
Ki
Li
Mu
Te
1
Te
2
Te
3
Te
4
Lu
Sp
Pr
TSSK-4
25x600bp-
600bp-
Mq
G
30x
+RT
-RT
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TSSK-4 mouse mRNATSSK-4 mouse mRNA
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Stage of expression TSSK-Stage of expression TSSK-
44
M 3 5 7 10
16
20
24
Mq
TSSK-4
25x
30x
600bp-
600bp-
3 5 7 10
16
20
24
Mq
M
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TSSK-4 ISHTSSK-4 ISH
Early + late spermatids and weakly late spermatocytes