8/8/2019 U19 Hope Lab June 2015

1/22

U19 conference call

 Thomas Hope labProject progress update

Katarina Kotnik Halavaty

 June 1! "1#

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2/22

$esults of the "nd group ofanimals

          •

   %  u  c   i   f  e  r  a  s  e

  a  c

   t   i  v   i   t  y

          •

   &   i  r  s   t   t  r  a  n  s   d  u  c  e   d

  c  e   l   l  s

          •

   P  r  o  v   i  r  a   l    '   (   )

   d  e   t  e  c

   t   i  o  n

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Uterusovaries

*ervi+

,agina

%o-er ,agina

Upper ,agina

)nalyses.

/nested P*/0uorescenmicroscopy

&reeing of tissue

2hole &$T

•  Tissue is soaked in luciferin for 1 minutes• 'etection of luciferase activity in &$T using an 3n ,ivo 3maging 4ystem

53,346

ssue uc erase act v tyscreen

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 T'& 3,$ 7 7 7 7 7 7 /

)nimals 889: 88#:# 88#;< 881:;< 88#"<

?vary - + ++ - - - -

Uterus - - - - - - -

*ervi+ - - - - - - -

,agina - - - - - - -

4ummary of luciferase activity

8/8/2019 U19 Hope Lab June 2015

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m*herry %uciferase@*A# Berge

m*herry *';@&3T* Berge

 Transduced cell in an ovary of acontrol pigtail macaCue

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m*herry@%uciferase merge

8/8/2019 U19 Hope Lab June 2015

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 T'& 3,$ 7 7 7 7 7 7 /

)nimals 889: 88#:# 88#;< 881:;< 88#"<

?vary 1 9 1 0 3

?vary "

4ummary of transduced cells inovaries

8/8/2019 U19 Hope Lab June 2015

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(o %T$ nested P*$ for PigtailBacaCues

1st P*$ product. 1># kb"nd P*$ product. "# bp

mCherry primers

1st P*$ product. # bp"nd P*$ product. ;# bp

LTR primers LTR primers

*B,%T$%uciferas

e3$D4 %T$m*herry

2P$D

 These animals -ere already challenged -ith 4H3,1"p; live virusE

8/8/2019 U19 Hope Lab June 2015

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1>

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 T'& 3,$ 7 7 7 7 7 7 /

)nimals 889: 88#:# 88#;< 881:;< 88#"<

?vary 1 - - - + - - +

?vary " - - - -

&allopian tubes

- + - -

*ervi+ - - + - + - -

,agina - - - - - - -

4ummary of proviral '() detection

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*onclusions

• %uciferase activity -as detected in. ovaries of t-o T'& 3,$ treated pigtail macaCues

• 4ingle infected cells -ere found in.

ovaries of t-o T'& 3,$ treated pigtail macaCues an ovary of a control animal

• Proviral '() -as detected by nested P*$ in.

 an ovary of T'& 3,$ treated animal an ovary of a control animal

*ervical tissue of t-o T'& 3,$ treated animals

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'ata of the :rd group ofanimals

   %  u

  c

   i   f  e  r

  a  s  e

  a  c   t   i

  v   i   t  y

Animal code88:19

88;"

88:1"

1>

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Preluciferin Postluciferin

!!319 "hole FRTPlacebo ring

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Postluciferin

!!319 #$aries

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Preluciferin Postluciferin

!!0%&' "hole FRT

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Postluciferin

!!0%&' #$aries

?variescut in half 

andreimaged

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Preluciferin Postluciferin

!!031' "hole FRT

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Postluciferin

!!031' #$aries

?varies cut in half and reimaged

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&allopian tubes

?varian fat

!!031'&allopian Tubes and ovarian fat

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Preluciferin Postluciferin

1(&))* "hole FRT

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1(&))* #$aries and Fallopian ,es

?varies&allopian tubes

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%uciferase activity summary

IVR + + + +

TDF - + + +

Animalcode

!!319 !!0%&' !!031' 1(&))*

#$aries + ++ + ++

.er,s - - - -

Cer$i/ - - - -

Vagina - - - -

Laia - - - -