Patent Application: US-201615234715-A

Abstract:
described herein are dna primer sequences designed for the determination of gene or transcript information from anuran species , and which may be used in studies for developmental and / or toxicity testing and for environmental toxicology or ecological assessment . also described herein is a rapid , sensitive , high - throughput assay useful for supporting potential risk assessment across vertebrate clades , and that is also useful for evaluation of complex contaminant mixtures .

Description:
the thyroid hormones ( ths ) are crucial for normal growth , development , and metabolism in all vertebrates including humans ( morreale de escobar , obregon et al . 2004 ; zoeller and rovett 2004 ). in fetal and neonatal mammals , environmental contaminants such as perchlorate and polychlorinated biphenyls ( pcbs ) impact th action contributing to serious defects in development and organ function including cognitive and motor deficits , abnormal bone growth , cardiac problems , and altered lipid metabolism ( zoeller and rovett 2004 ; bernal 2005 ; cheng 2005 ; siesser , cheng et al . 2005 ; liu and brent 2010 ). the brain is particularly vulnerable ( porterfield 1994 ; brucker - davis 1998 ; jones , thoemke et al . 2005 ; zoeller 2005 ; zoeller and crofton 2005 ) and the impact of mild hypothyroidism during critical developmental phases can have significant societal costs ( miller , crofton et al . 2009 ). in amphibians , ths are required for tadpole metamorphosis into a juvenile frog and disruption of th action results in the inability of the frog to develop normally ( shi 2000 ). therefore , modification of th action at early life history stages can lead to dramatic , deleterious outcomes for both humans and wildlife species . the mechanisms of th action are common among vertebrates and involve a complex regulatory pathway yet the high degree of sensitivity to th disruption of the tadpole has been identified as a powerful surrogate for the identification of chemicals and / or contaminants that perturb th signaling in vertebrates ( oecd 2009 ; helbing 2012 ). the invention presents an innovative method that relies upon a gene expression readout for detecting th disrupting activities using anuran species as model organisms , thereby greatly improving the power of the frog tadpole metamorphosis assay in predictivity and applicability to both laboratory testing and field applications across a range of all frog sentinel species . moreover , the invention describes validated dna primer sets for genes that are essential as references or “ normalizers .” these normalizers can be used alone or in conjunction with primers used to ascertain th responsiveness increasing the breadth of utility and application . furthermore , this set of normalizer genes is not just specific for thyroid function , but will also serve as a strong suite of reference genes for any type of qpcr application utilizing any response genes evaluated for any study using anuran species . the invention comprises one or more dna primer pairs ( table 1 ) that have been designed against “ normalizer ” and / or responsive genes such that the same primer pair representing a given gene can reliably and quantitatively function in anura regardless of species . examples of the use of the invention are to indicate responsiveness to thyroid hormone action or indicators of developmental phases attained . we demonstrate that these primers function well across the evolutionary range of anurans and also demonstrate the range of tissues in which the primers may be reliably used in the context of assessing mrna ( cdna ) levels or gene levels ( genomic dna ). finally , we also present a specific application of the technology to identifying perturbations in thyroid hormone signaling . a great diversity of anuran species are used in a broad range of disciplines such as developmental and cancer biology , physiology , ecology , molecular biology , biochemistry , from egg to adult stages . every qpcr - based study requires the use of reference gene transcripts or genes in order to be able to interpret experimental data . the basic assumption is that the reference gene ( s ) do not vary due to the treatment condition of the experiment and the type of reference or “ normalizer ” varies depending upon experimental context ( bustin , beaulieu et al . 2010 ). therefore , a selection of normalizer gene candidates is necessary as well as the identification and use of multiple normalizers to enhance the robustness of the data set . furthermore , this set of normalizer genes is not just specific for thyroid function , but will also serve as a strong suite of reference genes for any type of qpcr application utilizing any response genes evaluated for any study using anuran species . the inventors have identified th - responsive gene candidates that are common between mammals and frogs that would serve to act as meaningful indicators of th disruption within the context of an amphibian metamorphosis assay ( ama ) ( searcy , beckstrom - sternberg et al . 2012 ). the suggested species for the ama is xenopus laevis with additional interest in using the related silurana ( xenopus ) tropicalis ( mitsui , fujii et al . 2006 ). however , several nations are also interested in using native species relevant to their environments such as rana pipiens and pseudacris regilla ( canada ), rana temporaria ( europe ), and rana rugosa ( japan ), rhinella ( bufo ) marinus ( latin america , australia and se asia ), or the only truly cosmopolitan frog , rana catesbeiana ( bruno 2001 ; veldhoen , skirrow et al . 2006 ; oka , miyahara et al . 2009 ; marlatt , veldhoen et al . 2013 ). toxicological evaluations on a variety of species is highly desirable to enable direct comparison between species for sensitivities and evaluation of the impact of different life histories and genome compositions ( relyea and jones 2009 ; helbing 2012 ). the designed primers were subjected to tests of qpcr specificity and suitability within each tissue of interest in both xenopus laevis and rana catesbeiana . these two species bracket the most commonly used frog families spanning ˜ 200 million years of evolution ( sumida , kato et al . 2004 ) ( fig1 ). the inventors determined that if the primer pairs work well for both species , then the likelihood that they work for intermediate species is assured . fig1 is an anuran cladogram depicting major families of current interest in the scientific and conservation realm . xenopus laevis is a member of the pipidae whereas rana catesbeiana is a member of the ranidae . adapted from amphibiaweb : phylogenetic view of amphibia [ web application ] 2012 . uc regents , berkely , calif ., available : http : amphibiaweb . org ( 14 may 2012 ). the inventors &# 39 ; rigorous design and verification protocol includes amplicon sequence confirmation , routine melting curve analysis , generation of standard curves for each primer set , and determination of amplification efficiency . all procedures are compliant with the minimum information for publication of qpcr experiments ( miqe précis ) guidelines ( bustin , beaulieu et al . 2010 ). qpcr technology presents the most accurate and sensitive genetic technique available that enables high - throughput detection of low abundance mrna transcripts . due to its relative robustness and low operating cost , this technology is currently the most conducive to the demands of the regulatory context and the assays developed have immediate translation to regulatory labs . we elected to focus on a sybr - based detection method because this is the presently least expensive to run ( good for the price point of the end user ) and more labs have access to the equipment necessary for readout therefore increasing the market potential . our primer quality assurance / control procedure has been standardized and follows a three - tiered quality control and quality assurance ( qc / qa ) process , as follows . total rna from the target animal species and tissue type is converted to complementary dna ( cdna ) and queried with the putative qpcr primer pairs designed against genes of interest . the function of each primer pair in the qpcr assay is assessed for amplification signal representative of a single targeted cdna product . control qpcr reactions containing no cdna are performed to evaluate background inter - and intra - primer interactions that can contribute to signal noise . thermocycle conditions are chosen for each gene - specific primer pair that maximizes signal specificity while minimizing background noise . the dna products generated in the qpcr assay are subsequently confirmed to be the correct predicted length through agarose gel electrophoretic analysis . the specificity in dna amplification of each qpcr primer pair is further confirmed through isolation and sequence - dependent assessment of qpcr - generated dna products that contribute to the sybr - based signal . two methods can be used : 1 ) mapping of restriction endonuclease - derived fragments of qpcr amplified dna , or 2 ) dna sequencing of the product of qpcr . this serves to confirm the specificity of the qpcr sybr - signal collected and the identity of the gene sequence amplified by each primer pair used in qpcr . to ensure that the sybr - based signal data collected during qpcr can be employed in a quantitative manner , similarity in relative primer pair performance must be satisfied using information obtained through qpcr analysis of a dilution series of target cdna from the tissue of interest . the sensitivity and ability of each gene - specific primer pair to detect linear changes in target cdna abundance is assessed . primer pair performance of a test gene is compared to the performance of primer pairs from reference genes to ensure comparable amplification efficiencies . tissue context is important because we and others have found that some primers , although specific for their gene target , can display reduced performance in certain tissue contexts . the results of the three tier qc / qa process for each of the primer sets listed in table 1 are presented in fig2 . this demonstrates that the primer sets work to specifically amplify the appropriate amplicon in a variety of contexts . a summary of which tissue types are usable for transcriptomics analyses for each primer set is found in fig8 . in fig8 , the particular tissues of rana catesbeiana and xenopus laevis in which each primer set is usable are denoted as follows : the qc / qa results plus evidence of functionality in rana catesbeiana and xenopus laevis tissues are presented in fig2 , which is comprised of fig2 a ( 1 ) through fig2 a ( 10 ); fig2 b ( 1 ) through fig2 b ( 10 ) and fig2 c ( 1 ) through fig2 c ( 10 ). in these figures , results of the three tier qc / qa process by primer set on tissues from rana catesbeiana and xenopus laevis are shown . each of the 30 primer sets is identified at the top of each drawing figure . quality assurance ( qa ) of each primer pair is established through a three - tier evaluation procedure that includes : tier 1 , a clean cdna - dependent signal return with low background noise determined through amplification that is associated with the production of a single dna product for each anuran tissue type as determined through dissociation curves and electrophoretic analyses ; tier 2 , sequence - dependent confirmation of gene - specific targeting through restriction endonuclease mapping or direct dna sequencing of the isolated amplified dna product ; and tier 3 , primer pair comparative efficiency assessment across a tissue - specific cdna dilution series as it related to the qpcr performance of the selected reference primer pair on the same cdna dilution series . the slope of the interrelated qpcr performance must be ≦ 0 . 1 to be considered suitable for that tissue type . the decision of whether a primer pair would be used to discern a gene transcript as a normalizer or test is highly dependent upon the experimental context and includes considerations of tissue - type , sample time , exposure type , and species examined . examples of normalizer gene candidates that would be used in any qpcr experiment to act as input reference include , but are not limited to : col1a2 , actb , fn1 , rps10 , plp1 , rpl8 , eef1a1 and timm50 ( table 1 ). examples of thyroid hormone - responsive gene candidates in rana catesbeiana ( fig3 and 4 ) and xenopus laevis ( fig5 ) include , but are not limited to : col1a2 , actb , plp1 , thra , thrb , dio2 , thibz , klf9 , fn1 , mmp2 , and otc . fig3 illustrates a demonstration of thyroid hormone responsiveness of the target gene transcripts in rana catesbeiana . fold - change expression data for the indicated gene transcripts as determined using the comparative ct method ( δδct ) using multiple gene transcript normalizers . data are expressed in box plots as fold - change in expression levels relative to the vehicle control group . premetamorphic tadpoles were injected with either vehicle control ( c ) or the indicated concentration of thyroid hormone and the tissues collected after 48 hours . the concentration for t 3 exposures are 0 . 01 , 0 . 1 , 1 . 0 , 10 , 25 , and 50 nm and 0 . 05 , 0 . 5 , 5 . 0 , 50 , 125 , and 250 nm for t 4 . the medians are shown as solid black lines within the box and the box indicates the 25 th and 75 th percentiles . whiskers indicated the range . “ a ”: significantly different from the vehicle control group ( p & lt ; 0 . 05 ). “ n / a ” means not expressed in that tissue . fig4 illustrates a demonstration of thyroid hormone responsiveness of the target gene transcripts in rana catesbeiana . fold - change expression data for the indicated gene transcripts as determined using the comparative ct method ( δδct ) using multiple gene transcript normalizers . data are expressed in box plots as fold - change in expression levels relative to the vehicle control group . premetamorphic tadpoles were injected with either vehicle control ( c ) or the indicated concentration of thyroid hormone and the tissues collected after 48 hours . the concentration for t 3 exposures are 0 . 01 , 0 . 1 , 1 . 0 , 10 , 25 , and 50 nm and 0 . 05 , 0 . 5 , 5 . 0 , 50 , 125 , and 250 nm for t 4 . the medians are shown as solid black lines within the box and the box indicates the 25 th and 75 th percentiles . whiskers indicated the range . “ a ”: significantly different from the vehicle control group ( p & lt ; 0 . 05 ). “ n / a ” means not expressed in that tissue . fig5 illustrates a demonstration of thyroid hormone responsiveness of the target gene transcripts in xenopus laevis in the tail , brain , liver , and intestine . fold - change expression data for the indicated gene transcripts as determined using the comparative ct method ( δδct ) using multiple gene transcript normalizers . data are expressed in box plots as fold - change in expression levels relative to the vehicle control group . premetamorphic tadpoles were immersed in water containing vehicle control of 10 nm t 3 for 48 hours . the medians are shown as solid black lines within the box and the box indicates the 25 th and 75 th percentiles . whiskers indicate the range . greyed out regions indicate no detectable specific signal in the indicated tissue ( refer to fig8 for details ). “ a ”: signficantly different from the vehicle control group ( p & lt ; 0 . 05 ). the primer sets can be used on multiple anuran species beyond rana catesbeiana and xenopus laevis , as is demonstrated in fig6 . fig6 illustrates a demonstration that the primer sets are usable in a wide range of anuran specifies . the primer sets in table 1 were run on cdna preparations made from a single tissue or mixture of tissues ( species - dependent ) on the indicated species to demonstrate that the appropriately sized amplicon is produced across a wide range of anuran species . another use of the primers can be for detection of contaminating genomic dna in transcriptomic studies or in amplification of genomic dna ( fig7 ) and their utility in rana catesbeiana and xenopus laevis are summarized in table 2 . fig7 illustrates representative examples of primer set performance on genomic dna across different anuran species . genomic dna ( gdna ) was isolated from skin of xenopus laevis tadpoles and liver of rana catesbeiana adults . for both species , complementary dna ( cdna ) was generated from total rna isolated from mixed prometamorphic tadpole tissues . location of the predicted pcr amplicon representative of expressed sequence for each gene target is shown by an arrowhead . mw denotes molecular size standards in kilobase pairs of linear dna . the results for all of the primer sets are summarized in table 2 . each primer pair can be used independently of each other . in one embodiment , the invention comprises use of a primer pair by itself to detect particular genes within an anuran nucleic acid sample . in another embodiment , two or more of any of the primer pairs listed in table 1 may be used . the normalizer gene panel would include two or more of the following primer pairs : actb , rps10 , rpl8 , and eef1a1 . the end - user would then be able to use evaluation tools readily available to finalize the appropriateness of each reference gene . the thyroid hormone responsive gene panel would include two or more of the following primer pairs : col1a2 , plp1 , thra , thrb , dio2 , thibz , klf9 , fn1 , mmp2 , otc and timm50 . the assay of the invention further comprises the following method . tadpoles are exposed to the compound of interest in water and , at the appropriate time ( e . g ., within hours to a few days ), two standardized portions of the tail are removed . the tail tips are immediately subjected to an apoptosis assay in phase 1 , while the other ( second ) tail portion is preserved for phase 2 . if the results for phase 1 are negative , then the substance is not thyroid axis active . no further analysis is necessary . if the phase 1 results are positive , then the second tail portion is analyzed for shifts in the quantity of mrna transcripts specifically identified for responsiveness in phase 2 . an example of phase 2 analysis is using quantitative real time polymerase chain reaction ( qpcr ) for each gene transcript target . if the gene transcripts indicative of thyroid hormone ( th ) action are not affected , then it can be concluded that the test compound is not thyroid axis active . if the transcripts are affected , then the compound is identified as a likely th disruptor . accordingly , disclosed herein is a frog tadpole metamorphosis assay method comprising a first phase and a second phase , wherein : the first phase comprises exposing a tadpole to a compound of interest , removing first and second portions of the tadpole &# 39 ; s tail , subjecting the first portion to an apatosis assay , and the second phase comprises subjecting the second portion to an assay to detect shifts in a quantity of pre - determined mrna transcripts . in a more specific embodiment of the method , the first phase further comprises determining whether the compound of interest is thyroid axis active . in yet another embodiment of the method , the second phase further comprises subjecting the second portion to qpcr . in still yet another embodiment , the first phase further comprises determining whether the compound of interest is thyroid axis active , and the second phase further comprises subjecting the second portion to qpcr . in an embodiment of the invention , disclosed is a metamorphosis assay wherein the first phase comprises exposing a tadpole to a compound of interest , removing first and second portions of the tadpole &# 39 ; s tail , and subjecting the first portion to an apatosis assay , and the second phase comprises subjecting the second portion to qpcr using a primer pair comprising a first primer and a second primer , wherein the primer pair is selected from the group consisting of primer pairs a , b , c , d , e , f , g , h , i , j , k , l , m , n , and o : in yet another particular embodiment of the invention , the second phase further comprises subjecting the second portion to qpcr using two or more primer pairs , wherein each pair comprises a first primer and a second primer , and the primer pairs are selected from the group consisting of primer pairs a , b , d , e , f , g , h , i , j , k , and o : the invention further relates to a composition of matter comprising a primer pair selected from the group consisting of primer pairs a , b , c , d , e , f , g , h , i , j , k , l , m , n , and o : yet another aspect of the invention relates to a normalizer gene panel comprising two or more primer pairs selected from the group consisting of primer pairs b , c , l , and m : yet another aspect of the invention relates to a thyroid hormone responsive gene panel comprising two or more primer pairs selected from the group consisting of primer pairs a , d , e , f , g , h , i , j , k , n and o : amphibiaweb . 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