all procedures for the animal experiment were reviewed and approved by the animal care 
 and use committee of the national institute for physiological sciences , okazaki , japan . 

specific pathogen - free rats were purchased from charles river laboratories japan 
 ( kanagawa , japan ) .
all rats were housed in an environmentally controlled room with a 12-h 
 dark/12-h light cycle at a temperature of 23  2 c and humidity of 55  5% and were given 
 free access to a laboratory diet ( ce-2 ; clea japan , tokyo , japan ) and filtered water . 
 black coat color was used to identify wdb ( a rat strain  blk  which we described in 
 previous papers [ 14 , 20 , 21 ] has been renamed 
  wdb / nips  and registered to rat genome database , http://rgd.mcw.edu/wg/home , with the rgd 
 i d 7411634 ) . a homozygous cag / venus transgenic rat strain , generated with the crlj : wi 
 background ,
was named  cag / venus tg ( wi ) .  a 
 knock - in rat strain , generated with es cells originating from the wdb strain , was named 
scale bars : 100 m . ) were harvested and freed from their zonae pellucidae .
2b ) were cultured for 7 days in ( 1 ) 2i 
 medium consisting of 1 m mek inhibitor ( pd0325901 ; stemgent , cambridge , ma , usa ) and 3 m 
 gsk3 inhibitor ( chir99021 ; axon medchem bv , groningen , netherlands ) in n2b27 medium , ( 2 ) 2i medium with 1,000 u / ml rat lif 
 ( esgro ; millipore , bedford , ma , usa ) , ( 3 ) 2i medium with 10 m forskolin 
 ( sigma - aldrich , st .
louis , mo , usa ) , and ( 4 ) 2i medium with 1,000 u / ml rat lif and 10 m 
 forskolin on a feeder layer of mitomycin c - treated mouse embryonic fibroblasts .
2c ) were 
 disaggregated and transferred to new culture vessels containing the same culture medium 
 ( passage-1 ; p1 ) . these tentative es cell lines ( fig . 

2d ) were maintained by medium exchange every other day and 
 trypsinization / expansion every three days ( p2 plus ) .
gender of the es cell lines was 
 determined by pcr amplification of the rat y chromosome - specific sry gene using a 
 redextract - n - amp kit ( sigma - aldrich ) .
a 104-bp fragment of the rat sry gene was amplified 
 by 1 cycle for 3 min at 94 c ; 38 cycles for 30 sec at 94 c , 30 sec at 55 c and 30 sec at 
 72 c ; and 1 cycle for 10 min at 72 c with a primer pair of sry - f ( 5-cat cga agg gtt aaa 
 gtg cca-3 ) and sry - r ( 5-ata gtg tgt agg ttg ttg tcc-3 ) .  
the expression of stem cell marker genes ( oct-4 , rnanog , fgf-4 and rex-1 ) , a 
 trophectoderm - specific marker gene ( cdx2 ) and reference genes ( gata6 and -actin ) was 
 examined by rt - pcr analysis .
the primer sets used were the same as those described in our 
 recent publication .
briefly , total rna was 
 extracted from each sample using a rneasy mini kit ( qiagen , germantown , md , 
 usa ) . the cdna was prepared using a superscript iii first - strand synthesis system 
 ( invitrogen , carlsbad , ca , usa ) and amplified with takara la taq ( takara bio , 
 shiga , japan ) by 30 cycles at 95 c for 30 sec , at 55 c ( or 60 c in case of rnanog and 
 gata6 ) for 30 sec and at 72 c for 60 sec .
the potential of each es cell line for germline transmission was examined by the 
 conventional approach using g1 generation offspring via chimeric rats .
chimeric rats were prepared by microinjection of 
 1015 cells from each es cell line at p5 - 19 into the blastocoel cavity of wi e4.5 
 blastocysts , as described previously .
when the 
 albino wi - derived es cells ( without fluorescent markers ) were used , the host blastocysts 
 were from the wdb female rats .
all chimeric male rats that could develop to adulthood were 
 used for the progeny test .
contribution of the es cells in the resultant offspring was 
 confirmed by their coat color .
when germline - competent g1 offspring were not obtained , the 
 progeny test was terminated with the negative data from at least 5 chimeric rats per es 
 cell line and 4 litters per chimeric rat .
data regarding the outgrowth rate and the establishment efficiency at p5 were analyzed by 
 fisher s exact probability test using js - star 2012 , a program available online 
 ( http://www.physics.csbsju.edu/stats/index.html ) .
percentage data regarding the full - term 
 development of es cell - injected blastocysts , chimeric rat production and 
 germline - competent chimeras were arcsine transformed and analyzed by fisher s least 
 significant difference test after one - way anova .
all procedures for the animal experiment were reviewed and approved by the animal care 
 and use committee of the national institute for physiological sciences , okazaki , japan . 

specific pathogen - free rats were purchased from charles river laboratories japan 
 ( kanagawa , japan ) .
all rats were housed in an environmentally controlled room with a 12-h 
 dark/12-h light cycle at a temperature of 23  2 c and humidity of 55  5% and were given 
 free access to a laboratory diet ( ce-2 ; clea japan , tokyo , japan ) and filtered water . 
 black coat color was used to identify wdb ( a rat strain  blk  which we described in 
 previous papers [ 14 , 20 , 21 ] has been renamed 
  wdb / nips  and registered to rat genome database , http://rgd.mcw.edu/wg/home , with the rgd 
 i d 7411634 ) . a homozygous cag / venus transgenic rat strain , generated with the crlj : wi 
 background ,
was named  cag / venus tg ( wi ) .  a 
 knock - in rat strain , generated with es cells originating from the wdb strain , was named 
scale bars : 100 m . ) were harvested and freed from their zonae pellucidae .
2b ) were cultured for 7 days in ( 1 ) 2i 
 medium consisting of 1 m mek inhibitor ( pd0325901 ; stemgent , cambridge , ma , usa ) and 3 m 
 gsk3 inhibitor ( chir99021 ; axon medchem bv , groningen , netherlands ) in n2b27 medium , ( 2 ) 2i medium with 1,000 u / ml rat lif 
 ( esgro ; millipore , bedford , ma , usa ) , ( 3 ) 2i medium with 10 m forskolin 
 ( sigma - aldrich , st .
louis , mo , usa ) , and ( 4 ) 2i medium with 1,000 u / ml rat lif and 10 m 
 forskolin on a feeder layer of mitomycin c - treated mouse embryonic fibroblasts .
2c ) were 
 disaggregated and transferred to new culture vessels containing the same culture medium 
 ( passage-1 ; p1 ) . these tentative es cell lines ( fig . 

2d ) were maintained by medium exchange every other day and 
 trypsinization / expansion every three days ( p2 plus ) .
gender of the es cell lines was 
 determined by pcr amplification of the rat y chromosome - specific sry gene using a 
 redextract - n - amp kit ( sigma - aldrich ) .
a 104-bp fragment of the rat sry gene was amplified 
 by 1 cycle for 3 min at 94 c ; 38 cycles for 30 sec at 94 c , 30 sec at 55 c and 30 sec at 
 72 c ; and 1 cycle for 10 min at 72 c with a primer pair of sry - f ( 5-cat cga agg gtt aaa 
 gtg cca-3 ) and sry - r ( 5-ata gtg tgt agg ttg ttg tcc-3 ) .  
the expression of stem cell marker genes ( oct-4 , rnanog , fgf-4 and rex-1 ) , a 
 trophectoderm - specific marker gene ( cdx2 ) and reference genes ( gata6 and -actin ) was 
 examined by rt - pcr analysis .
the primer sets used were the same as those described in our 
 recent publication .
briefly , total rna was 
 extracted from each sample using a rneasy mini kit ( qiagen , germantown , md , 
 usa ) .
the cdna was prepared using a superscript iii first - strand synthesis system 
 ( invitrogen , carlsbad , ca , usa ) and amplified with takara la taq ( takara bio , 
 shiga , japan ) by 30 cycles at 95 c for 30 sec , at 55 c ( or 60 c in case of rnanog and 
 gata6 ) for 30 sec and at 72 c for 60 sec .
the potential of each es cell line for germline transmission was examined by the 
 conventional approach using g1 generation offspring via chimeric rats .
chimeric rats were prepared by microinjection of 
 1015 cells from each es cell line at p5 - 19 into the blastocoel cavity of wi e4.5 
 blastocysts , as described previously .
when the 
 albino wi - derived es cells ( without fluorescent markers ) were used , the host blastocysts 
 were from the wdb female rats .
all chimeric male rats that could develop to adulthood were 
 used for the progeny test .
contribution of the es cells in the resultant offspring was 
 confirmed by their coat color .
when germline - competent g1 offspring were not obtained , the 
 progeny test was terminated with the negative data from at least 5 chimeric rats per es 
 cell line and 4 litters per chimeric rat .
data regarding the outgrowth rate and the establishment efficiency at p5 were analyzed by 
 fisher s exact probability test using js - star 2012 , a program available online 
 ( http://www.physics.csbsju.edu/stats/index.html ) .
percentage data regarding the full - term 
 development of es cell - injected blastocysts , chimeric rat production and 
 germline - competent chimeras were arcsine transformed and analyzed by fisher s least 
 significant difference test after one - way anova .
 abstract this study was designed to investigate whether supplementation of 2i medium with 
 leukemia inhibitory factor ( lif ) and/or forskolin would support establishment of 
 germline - competent rat embryonic stem ( es ) cell lines . due to the higher likelihood of 
 outgrowth rates , supplementation of forskolin with or without lif contributed to the 
 higher establishment efficiency of es cell lines in the wdb strain . 
 germline transmission 
 competency of the chimeric rats was not influenced by the profile of es cell lines until 
 their establishment . 
 when the lif / forskolin - supplemented 2i medium was used , the rat 
 strain used as the blastocyst donor , such as the wi strain , was a possible factor 
 negatively influencing the establishment efficiency of es cell lines . 
 once es cell lines 
 were established , all lines were found to be germline - competent by a progeny test in 
 chimeric rats . in conclusion , both lif and forskolin are not essential but can play a 
 beneficial role in the establishment of  genuine  rat es cell lines . 
Methods
Animals
Establishment of ES cells
Expression of stem cell marker genes
Germline transmission
Statistics
Supplementary