BACKGROUND
chick embryos have long been a favorite of developmental biologists, in large part because they are amenable to in ovo experimentation.
however, until recently the chick has lacked a robust lost-of-function technique for studies of gene function.
chick gene "knock outs" are impractical because es cells are elusive, flocks are unwieldy, and generation times are long  <cit> .
chick loss-of-function studies, as a result, have resorted to indirect methods such as dominant negative alleles   <cit> .
other approaches have included the use of chemical inhibitors to reduce gene function, such as cyclopamine for hedgehog action and su <dig> for fibroblast growth factor signaling  <cit> .
more recently, rna interference  techniques have been used successfully in various chick tissues  <cit> .
chick embryos electroporated with small interfering rnas  show knock down of the targeted gene.
however, electroporation techniques are transient, because the effects disappear once cells lose the introduced nucleic acid.
another more stable delivery method uses the replication competent avian splice  retroviruses to introduce hairpins into tissues.
our group and others have used such vectors to reduce gene function in a variety of tissues, including developing craniofacial tissues, the limb bud, and the dorsal root ganglion  <cit> .
in this report, we demonstrate that the rcas-rnai technique works successfully in the developing chick retina.
we show that this loss-of-function method offers distinct advantages.
it is long lasting, because the viral vector integrates stably into the genome of infected retinal cells.
it is also transmissible, because infected retinal cells release more virus that spread to neighboring cells.
finally, it is easily traced, because each infected cell is clearly marked by viral antigens.
this technique thus leads to large patches of infected retinal tissue with knock down of a targeted gene.
RESULTS
rna interference occurs in chick retinal cells in vitro
the first goal was to establish that rnai can work in chick retinal cells.
a dna-based rnai strategy previously shown to produce small hairpins  effective in mammalian cells was used   <cit> .
the mouse u <dig> promoter uses rna polymerase iii to transcribe shrnas, which are then processed into intermediates that knock down a targeted gene.
an rna polymerase iii promoter is used because rna polymerase iii efficiently transcribes short transcripts and then terminates efficiently.
a major question was whether the mouse u <dig> promoter would be compatible with the transcriptional machinery in the chick retina.
a diagram of the bs/u <dig> vector, showing the murine rna polymerase iii-specific u <dig> promoter, the location for oligonucleotide cloning which code for the hairpin, and the  <dig> consecutive thymidine bases which serve as a termination sequence for rna polymerase iii.
portions of the visinin sequence used to make hairpins.
four sequences depicted in red were chosen from the coding sequence.
each sequence started with three guanine bases, and each sequence was  <dig> nucleotides long.
oligonucleotides coding for these sequences were cloned into the bs/u <dig> vector as described in the materials and methods.
electroporation of chick retinas with bs/u6-vis constructs in vitro.
e <dig> retinas were electroporated with each construct and a gfp construct, and then were cultured for two days.
retinas were then dissociated and gfp positive cells were scored for visinin expression.
visinin  was chosen as the test gene for rnai function in the chick retina.
visinin, a calcium-binding protein expressed in photoreceptors, has distinct advantages as a test gene  <cit> .
the visinin transcript is abundant and there are anti-visinin monoclonal and polyclonal antibodies.
furthermore, visinin reduction should not lead to drastic phenotypes that could confound an assessment of the technique.
this prediction is based on the observation that mouse knock outs of recoverin, a mammalian calcium binding protein in photoreceptor cells, only leads to subtle defects in phototransduction  <cit> .
hairpins were designed against four different regions of the visinin coding sequence.
oligonucleotides coding for the hairpins were then cloned into the pbs/u <dig> vector.
the shrna plasmids were co-electroporated with a gfp-expressing plasmid into the explants of embryonic day  <dig>  retinas.
the retinas were then cultured for  <dig> days, dissociated, and assayed for the number of gfp-positive, visinin-positive cells.
in these experiments, gfp was scored by intrinsic gfp fluorescence and visinin was detected with a monoclonal antibody .
constructs with hairpin  <dig> or hairpin  <dig> produced the same number of gfp-positive, visinin-positive cells as control hairpin transfected retina, while hairpins  <dig> and  <dig> significantly reduced the number of double-positive cells.
these results suggest that hairpin  <dig> and  <dig> can reduce the amount of visinin protein.
rcas viruses can deliver rnai into the chick retina in vivo
electroporation leads only to transient association of dna with transduced cells and only in limited areas.
for a more stable effect that can cover a wide area, the rcas vector was used to deliver the u6-hairpin.
rcas virus would allow infected cells to continually express the hairpin, because the virus integrates into the cellular genome.
furthermore, these viruses are replication-competent and spread throughout the retina, resulting in patches of infected tissue or a completely infected retina  <cit> .
there were two potential complications with an rcas-u <dig> hairpin virus.
one possible problem is that it contains two promoters, a 5' ltr recognized by rna polymerase ii, which is essential for proper viral replication, and the murine u <dig> promoter for transcription of the hairpins.
it was unclear whether these two promoters would interfere with one another.
the second potential problem was that the viral genome and sub-genomic spliced mrnas might be targeted for degradation, thus reducing viral titers and possibly leading to selection of genomes with the hairpin or u <dig> promoter deleted.
to determine empirically if there was an optimum arrangement of the u <dig> promoter and the hairpin sequence orientation with respect to the ltr and the viral genome, two rcas constructs were made.
one placed the 5'ltr and u <dig> promoter in the same orientation.
the second arranged the two promoters such that they directed transcription in opposite directions.
four different viruses were made using this strategy and hairpins  <dig> and 2: rcas-u6-vis1forward, rcas-u6-vis1reverse, rcas-u6-vis2forward, and rcas-u6-vis2reverse .
all  <dig> viruses grew to normal titers , indicating that the viral genome was not successfully targeted by the hairpins that they encode.
when the viruses were introduced into the retina, both rcas-u6-vis <dig> viruses did not reduce visinin expression .
infected whole mount retinas showed normal visinin expression throughout the retina, with lighter staining in a center spot and equatorial stripe.
these results agree with the culture results, in which hairpin  <dig> did not reduce the number of visinin-positive cells.
also in agreement with the culture results, both rcas-u6-vis <dig> viruses reduced visinin expression in areas of infection.
the knock down was significant, though not complete, perhaps reflecting the efficiency of rnai.
furthermore, knock down occurred equally with the forward and reverse viruses, suggesting that promoter orientation does not affect viral rnai activity.
the area of knock down also could be manipulated by diluting the amount of virus injected .
to test whether the rcas-rnai vectors can be used more generally in the chick retina, other genes were tested, including the thyroid hormone processing enzyme deiodinase  <dig>    <cit> .
rcas-u6-dio <dig> viruses grew to normal titers, again suggesting that the rnai viruses do not target themselves.
dio <dig> activity was examined in infected eyes using an enzymatic assay.
the retinal pigmented epithelium  was assayed, as this tissue has high dio <dig> activity .
rpe infected with rcas-u6-vis <dig> exhibited strong dio <dig> activity, similar to uninfected tissue, demonstrating that the rcas hairpin does not non-specifically affect dio <dig>  in contrast, rpe infected with rcas-u6-dio <dig> had significantly lower levels of dio <dig> activity .
the fact that rcas-u6-dio <dig> infected rpe had some dio <dig> activity may reflect the difference between knock down and knock out, or may indicate that the rpe was not fully infected.
in addition, reduction of dio <dig> transcript in the retina was apparent by in situ hybridization .
the reduction correlated precisely with viral infection, and was specific to dio <dig> because rcas-u6-dio <dig> did not change visinin expression.
the results demonstrate that in situ hybridization is a convenient way to measure rcas-rnai effectiveness.
infection of chick retina with rcas-u6-dio <dig> and biochemical analysis of dio <dig> activity.
stage  <dig> retinas were injected with rcas-u6-vis <dig>  rcas-u6-dio <dig>  or left uninfected, and rpe tissue was harvested at approximately e <dig>   dio <dig> activity in the rpe was assayed by measuring free i <dig> production.
infection of chick retina with rcas-u6-dio <dig> and analysis of dio <dig> and visinin expression.
stage  <dig> retinas were injected with rcas-u6-dio <dig> and harvested at approximately e <dig>   dio <dig> and visinin transcript expression were assayed by in situ hybridization, followed by viral antigen detection using 3c <dig>  antibody.
CONCLUSIONS
we present a technique for gene knock down in the chick retina in vitro and in vivo, using rcas retroviruses to deliver hairpins mediating rnai.
the viruses were modified with the gateway sequences  to facilitate construction.
the viruses reduce both transcript and protein levels specific to their targeted gene.
furthermore, because rcas viruses integrate into the genome of infected cells as well as spread to neighboring dividing cells, rcas-rnai infection achieves knock down days after infection in large patches of tissue.
presently we are using the technique to study gene function of a variety of candidates in the developing retina.
furthermore, as we and others have shown, rcas-rnai should be useful for loss-of-function studies in any chick tissues accessible to retroviral infection  <cit> .
these data indicate that machinery mediating rnai is present in the chick retina, and that the murine u <dig> promoter is effective in the chick retina.
furthermore, the hairpins do not induce cell death, as evidenced by 1) the high viral titers obtained in chick fibroblast cells, 2) the large infected patches of retina, and 3) the normal tunel staining of infected tissue .
interestingly, because rcas-rnai viruses grow to normal titers, the viral genome appears protected from the hairpin that it encodes.
this may be due to a secondary structure that might form within the viral genome, as well as within the subgenomic spliced mrna that encodes the env protein.
if the target sequence is within a stable secondary structure, it may be inaccessible to the rnai effects of any transcribed hairpins.
an important caveat is that we have been unable to detect a reduction in rna levels by in situ hybridization on cryosectioned infected tissue.
cryosectioned tissue shows infection throughout the retinal thickness, in both photoreceptor and non-photoreceptor cells.
however, unlike whole mount in situ hybridization which easily detects reduced levels of the target transcript in infected patches, section in situ hybridization shows no detectable difference between infected and uninfected areas.
it is still unclear why this is the case, and may be related in part to the thickness of retinal sections.
perhaps if rnai led to complete absence, instead of knock down, section in situ hybridization would be a better method of screening for functional rcas-rnai constructs.

 chick embryos have long been a favorite of developmental biologists, in large part because they are amenable to in ovo experimentation. however, until recently the chick has lacked a robust lost-of-function technique for studies of gene function. chick gene "knock outs" are impractical because es cells are elusive, flocks are unwieldy, and generation times are long. chick loss-of-function studies, as a result, have resorted to indirect methods such as dominant negative alleles. other approaches have included the use of chemical inhibitors to reduce gene function, such as cyclopamine for hedgehog action and su for fibroblast growth factor signaling. more recently, rna interference techniques have been used successfully in various chick tissues. chick embryos electroporated with small interfering rnas show knock down of the targeted gene. however, electroporation techniques are transient, because the effects disappear once cells lose the introduced nucleic acid. another more stable delivery method uses the replication competent avian splice retroviruses to introduce hairpins into tissues. our group and others have used such vectors to reduce gene function in a variety of tissues, including developing craniofacial tissues, the limb bud, and the dorsal root ganglion. in this report, we demonstrate that the rcas-rnai technique works successfully in the developing chick retina. we show that this loss-of-function method offers distinct advantages. it is long lasting, because the viral vector integrates stably into the genome of infected retinal cells. it is also transmissible, because infected retinal cells release more virus that spread to neighboring cells. finally, it is easily traced, because each infected cell is clearly marked by viral antigens. this technique thus leads to large patches of infected retinal tissue with knock down of a targeted gene.  rna interference occurs in chick retinal cells in vitro the first goal was to establish that rnai can work in chick retinal cells. a dna-based rnai strategy previously shown to produce small hairpins effective in mammalian cells was used. the mouse u promoter uses rna polymerase iii to transcribe shrnas, which are then processed into intermediates that knock down a targeted gene. an rna polymerase iii promoter is used because rna polymerase iii efficiently transcribes short transcripts and then terminates efficiently. a major question was whether the mouse u promoter would be compatible with the transcriptional machinery in the chick retina. a diagram of the bs/u vector, showing the murine rna polymerase iii-specific u promoter, the location for oligonucleotide cloning which code for the hairpin, and the consecutive thymidine bases which serve as a termination sequence for rna polymerase iii. portions of the visinin sequence used to make hairpins. four sequences depicted in red were chosen from the coding sequence. each sequence started with three guanine bases, and each sequence was nucleotides long. oligonucleotides coding for these sequences were cloned into the bs/u vector as described in the materials and methods. electroporation of chick retinas with bs/u6-vis constructs in vitro. e retinas were electroporated with each construct and a gfp construct, and then were cultured for two days. retinas were then dissociated and gfp positive cells were scored for visinin expression. visinin was chosen as the test gene for rnai function in the chick retina. visinin, a calcium-binding protein expressed in photoreceptors, has distinct advantages as a test gene. the visinin transcript is abundant and there are anti-visinin monoclonal and polyclonal antibodies. furthermore, visinin reduction should not lead to drastic phenotypes that could confound an assessment of the technique. this prediction is based on the observation that mouse knock outs of recoverin, a mammalian calcium binding protein in photoreceptor cells, only leads to subtle defects in phototransduction. hairpins were designed against four different regions of the visinin coding sequence. oligonucleotides coding for the hairpins were then cloned into the pbs/u vector. the shrna plasmids were co-electroporated with a gfp-expressing plasmid into the explants of embryonic day retinas. the retinas were then cultured for days, dissociated, and assayed for the number of gfp-positive, visinin-positive cells. in these experiments, gfp was scored by intrinsic gfp fluorescence and visinin was detected with a monoclonal antibody. constructs with hairpin or hairpin produced the same number of gfp-positive, visinin-positive cells as control hairpin transfected retina, while hairpins and significantly reduced the number of double-positive cells. these results suggest that hairpin and can reduce the amount of visinin protein. rcas viruses can deliver rnai into the chick retina in vivo electroporation leads only to transient association of dna with transduced cells and only in limited areas. for a more stable effect that can cover a wide area, the rcas vector was used to deliver the u6-hairpin. rcas virus would allow infected cells to continually express the hairpin, because the virus integrates into the cellular genome. furthermore, these viruses are replication-competent and spread throughout the retina, resulting in patches of infected tissue or a completely infected retina. there were two potential complications with an rcas-u hairpin virus. one possible problem is that it contains two promoters, a 5' ltr recognized by rna polymerase ii, which is essential for proper viral replication, and the murine u promoter for transcription of the hairpins. it was unclear whether these two promoters would interfere with one another. the second potential problem was that the viral genome and sub-genomic spliced mrnas might be targeted for degradation, thus reducing viral titers and possibly leading to selection of genomes with the hairpin or u promoter deleted. to determine empirically if there was an optimum arrangement of the u promoter and the hairpin sequence orientation with respect to the ltr and the viral genome, two rcas constructs were made. one placed the 5'ltr and u promoter in the same orientation. the second arranged the two promoters such that they directed transcription in opposite directions. four different viruses were made using this strategy and hairpins and 2: rcas-u6-vis1forward, rcas-u6-vis1reverse, rcas-u6-vis2forward, and rcas-u6-vis2reverse. all viruses grew to normal titers, indicating that the viral genome was not successfully targeted by the hairpins that they encode. when the viruses were introduced into the retina, both rcas-u6-vis viruses did not reduce visinin expression. infected whole mount retinas showed normal visinin expression throughout the retina, with lighter staining in a center spot and equatorial stripe. these results agree with the culture results, in which hairpin did not reduce the number of visinin-positive cells. also in agreement with the culture results, both rcas-u6-vis viruses reduced visinin expression in areas of infection. the knock down was significant, though not complete, perhaps reflecting the efficiency of rnai. furthermore, knock down occurred equally with the forward and reverse viruses, suggesting that promoter orientation does not affect viral rnai activity. the area of knock down also could be manipulated by diluting the amount of virus injected. to test whether the rcas-rnai vectors can be used more generally in the chick retina, other genes were tested, including the thyroid hormone processing enzyme deiodinase. rcas-u6-dio viruses grew to normal titers, again suggesting that the rnai viruses do not target themselves. dio activity was examined in infected eyes using an enzymatic assay. the retinal pigmented epithelium was assayed, as this tissue has high dio activity. rpe infected with rcas-u6-vis exhibited strong dio activity, similar to uninfected tissue, demonstrating that the rcas hairpin does not non-specifically affect dio in contrast, rpe infected with rcas-u6-dio had significantly lower levels of dio activity. the fact that rcas-u6-dio infected rpe had some dio activity may reflect the difference between knock down and knock out, or may indicate that the rpe was not fully infected. in addition, reduction of dio transcript in the retina was apparent by in situ hybridization. the reduction correlated precisely with viral infection, and was specific to dio because rcas-u6-dio did not change visinin expression. the results demonstrate that in situ hybridization is a convenient way to measure rcas-rnai effectiveness. infection of chick retina with rcas-u6-dio and biochemical analysis of dio activity. stage retinas were injected with rcas-u6-vis rcas-u6-dio or left uninfected, and rpe tissue was harvested at approximately e dio activity in the rpe was assayed by measuring free i production. infection of chick retina with rcas-u6-dio and analysis of dio and visinin expression. stage retinas were injected with rcas-u6-dio and harvested at approximately e dio and visinin transcript expression were assayed by in situ hybridization, followed by viral antigen detection using 3c antibody.  we present a technique for gene knock down in the chick retina in vitro and in vivo, using rcas retroviruses to deliver hairpins mediating rnai. the viruses were modified with the gateway sequences to facilitate construction. the viruses reduce both transcript and protein levels specific to their targeted gene. furthermore, because rcas viruses integrate into the genome of infected cells as well as spread to neighboring dividing cells, rcas-rnai infection achieves knock down days after infection in large patches of tissue. presently we are using the technique to study gene function of a variety of candidates in the developing retina. furthermore, as we and others have shown, rcas-rnai should be useful for loss-of-function studies in any chick tissues accessible to retroviral infection. these data indicate that machinery mediating rnai is present in the chick retina, and that the murine u promoter is effective in the chick retina. furthermore, the hairpins do not induce cell death, as evidenced by 1) the high viral titers obtained in chick fibroblast cells, 2) the large infected patches of retina, and 3) the normal tunel staining of infected tissue. interestingly, because rcas-rnai viruses grow to normal titers, the viral genome appears protected from the hairpin that it encodes. this may be due to a secondary structure that might form within the viral genome, as well as within the subgenomic spliced mrna that encodes the env protein. if the target sequence is within a stable secondary structure, it may be inaccessible to the rnai effects of any transcribed hairpins. an important caveat is that we have been unable to detect a reduction in rna levels by in situ hybridization on cryosectioned infected tissue. cryosectioned tissue shows infection throughout the retinal thickness, in both photoreceptor and non-photoreceptor cells. however, unlike whole mount in situ hybridization which easily detects reduced levels of the target transcript in infected patches, section in situ hybridization shows no detectable difference between infected and uninfected areas. it is still unclear why this is the case, and may be related in part to the thickness of retinal sections. perhaps if rnai led to complete absence, instead of knock down, section in situ hybridization would be a better method of screening for functional rcas-rnai constructs. 
however, it has lacked an efficient loss-of-function method for studies of gene function. furthermore, the amount of retinal tissue affected can be controlled by manipulating the degree of infection. this technique provides a rapid and efficient loss-of-function option for studies in the developing chick retina. the knock down persists as the retina matures and can be detected using in situ hybridization.
 the embryonic chick provides an excellent model system for studies of development. however, it has lacked an efficient loss-of-function method for studies of gene function.  we show that avian retroviruses can deliver hairpins mediating rna interference to the developing chick eye. these viruses'knock down' specific genes in infected areas of the retina. the knock down persists as the retina matures and can be detected using in situ hybridization. furthermore, the amount of retinal tissue affected can be controlled by manipulating the degree of infection.  this technique provides a rapid and efficient loss-of-function option for studies in the developing chick retina. 