Patent Abstract:
disclosed is a transgenic knockout mouse whose genome has a homozygous disruption in its endogenous gpx1 and gpx2 genes , wherein the disruptions result in a decrease in gpx activity in the transgenic mice when compared to non transgenic mice of the same type . methods for production of the mouse are presented . also disclosed are cells derived from the transgenic knockout mouse . the mouse can be used in a method for identifying therapeutic agents for the treatment of an individual diagnosed with a metabolic disorder associated with a reduction or loss of expression of wild - type gpx1 and gpx2 .

Detailed Description:
the practice of the present invention employs , unless otherwise indicated , conventional techniques of chemistry , molecular biology , microbiology , recombinant dna , genetics , immunology , cell biology , cell culture and transgenic biology , which are known to one of ordinary skill in the related art . gpx1 - ko mice were generated by using standard techniques as c57bl6 / j ( 96 ) and 129sv / j hybrids and b6 inbred mice as described previously ( 11 ). the generation of gpx2 - ko mice as b6 and 129s3 hybrids and b6 mice has been described previously ( 13 ). these mice were housed in ventilated cage racks ( allentown caging equipment , allentown , n . j .) under conventional housing conditions . the colony was monitored for infectious pathogens via sentinel mouse surveillance and necropsy of randomly selected littermates of the double - ko mice . the loose stools of several double - ko mice were negative for parasites . all mice had free access to laboratory rodent diet ( 5001 , purina mills inc ., richmond , ind .) and water . this diet contains 23 % protein , 4 . 5 % fat , 6 % fiber , and 0 . 28 ppm selenium as provided by the manufacturer ( http :// www . labdiet . com ). genotyping of gpx1 and gpx2 - ko mice was done with either southern or pcr analysis of dna isolated from tails . for southern analysis , 10 μg dna was digested with bamhi or apai to determine the genotype of gpx1 and gpx2 , respectively . after overnight digestion , dna was resolved in 0 . 75 % agarose gel and transferred to zeta probe membrane ( biorad lab ., richmond , calif .) and probed with 32 p - labeled and random - primed 3 ′ ecori fragment of mouse gpx1 cdna and mouse gpx2 exon 2 cdna . the southern blot was analyzed by phosphor imaging ( molecular dynamics , sunnyvale , calif .) ( 13 ). polymerase chain reactions ( pcr ) were also performed . the pcr primers for the wildtype gpx1 allele were mpx101f ( dna seq id no . 1 : 5 ′- aaggaggtgcaggcggctgtgagcg - 3 ′) and gpx15 ( seq id no . 2 : 5 ′- accgttcaccttgcacttctc - 3 ′), which amplified about 600 bp dna fragment . the primers for the gpx1 - ko allele were ppntpgk ( seq id no . 3 : 5 ′- cagtttcatagcctgaagaacgagat - 3 ′) and gpx15 , which amplified a ˜ 200 bp dna fragment . the primers for the wildtype gpx2 allele were mpx206 ( seq id no . 4 : 5 ′- cccacctgtctagaggactta - 3 ′) and mpxin09 ( seq id no . 5 : 5 ′- tccatgccaacgtagtgatt - 3 ′), which amplified a ˜ 600 bp dna . the primers for the gpx2 - ko allele were mpx206 and ppntpgk , which amplified a ˜ 400 bp dna . both alleles were amplified in the same reaction tubes . rectal temperature was measured with thermalert mouse probe ( model th - 8 , physitemp instrument inc ., clifton , n . j .) at the 6 - 8 am on mice under normal housing . to quantify the food and water consumption and feces and urine output mice were placed in metabolic cages without bedding for 24 h . this setting appeared to be stressful for the double - ko mice , as shown by frequent hunched - over appearance , pilocrection of their coat , and loose stools the next day . mice were sacrificed by halothane overdose ( halocarbon labs , north augusta , s . c .). after removing the lumen contents , sections of jejunum , ileum , colon , and rectum were rinsed with phosphate buffered saline , and then fixed in 10 % buffered formalin or bouin &# 39 ; s fixative for 2 - 3 h . the tissues were then dehydrated in ethanol , and embedded in paraffin and sectioned onto slides . the tissue sections were stained with hematoxylin and eosin ( h & amp ; e ) alone or in addition to periodic acid schiff ( pas ) staining . gpx activity was determined on mouse intestinal and colon epithelium . jejunal and ileal epithelium were isolated from the proximal and the distal one third of small intestine as described previously ( 13 ). the gpx activity was measured with 60 μm h 2 o 2 and 3 mm gsh at ph 7 . 3 . the protein concentration was determined with a bca assay ( pierce chemical , rockford , ill .) with bovine serum albumin as the standard . the invention is further illustrated by the following examples , which are not intended to be limiting . homozygous gpx1 - ko and gpx2 - ko mice were bred to generate heterozygous double - ko mice . these heterozygous double - ko mice were bred to each other , one sixteenth of the offspring were homozygous double - ko mice . one half of mice were reciprocal homozygous and heterozygous ko &# 39 ; s , so called 3 - quarter ko &# 39 ; s . these double - ko and 3 - quarter ko were then used as breeders to generate double - ko mice . the genotypes of six mice were analyzed by southern analysis to examine genetic characteristics of the results of double knockout breeding . referring now to fig1 a , the left panel contains bamhi - digested dna hybridized with mouse gpx1 cdna . the top arrow points at ˜ 11 kb wildtype ( wt ) allele , and the lower arrow points at ˜ 4 . 3 kb gpx1 - ko allele . the right panel contains apai - digested dna hybridized with mouse gpx2 cdna . the top arrow points at ˜ 14 kb gpx2 pseudogene ( ps - gpx2 ), the middle arrow points at ˜ 7 kb wt allele , and the lowest arrow points at 4 . 9 kb gpx2 - ko allele . the other two dna fragments of low molecular weights do not correlate with gpx2 genotypes , and are ignored . the genotypes are shown in the bottom of the panels and are designated as follows :+/−, one wildtype and one knockout allele ; +/+ two wildtype alleles ; −/−, two knockout alleles . referring now to fig1 b , there are shown the results of gpx enzyme activity in the epithelium of mouse lower gi - tract . gpx activity was measured using hydrogen peroxide as the substrate . the error bars represent variances or standard deviations of the means . the number of mice assayed in each group from left to right is 3 , 2 , 4 , 4 , and 4 respectively . the genotypes for both gpx1 and gpx2 are as in 1a . the number of the double - ko mice was close to the predicted value from mendelian genetics . similar numbers of male and female offspring were obtained . this indicates that the double - ko mice have normal embryonic development and there is no gender bias . both male and female double - ko mice can be fertile but only a small percentage of mice gain enough weight and appear healthy enough to be used as breeders . referring now to fig2 a , there are shown the results of growth activity in adult ( 45 - 47 days old ) homozygous double - ko mice . there is shown a graphical representation of the growth rate of a single litter of 8 pups . male mice are shown in larger symbols , and female mice are shown in smaller symbols . circles represent gpx1 +/− gpx2 −/− mice , diamonds represent gpx1 −/− gpx2 +/− mice , squares represent gpx1 +/− gpx2 +/− mice , and triangles represent gpx1 −/− gpx2 −/− mice . the female and male double - ko mice in the top panel started to show growth retardation at 21 and 26 days old . referring now to fig2 b , there is shown a graphical representation of the number and age of 33 homozygous double - ko mice at which they first show growth retardation . the double - ko had almost background gpx activity in the mucosa of small and large intestine ( lower panel of fig1 ). since the jejunum mucosa had a high level of gpx - 1 and low level of gpx - gi as shown previously ( 4 ), the total gpx activity in this region corresponded only to the gpx1 gene dosage . the gpx - gi contributed little to gpx activity in the jejunum even in a homozygous gpx1 - ko background since the heterozygous and homozygous gpx2 - ko mice do not have statistically different gpx activity ( p = 0 . 10 ) as shown in the last two groups in fig1 a . a lower level of gpx - 1 and a higher level of gpx - gi are expressed in the ileal mucosa compared with that in jejunal mucosa . the dosage effect of the gpx2 allele is evident only in the absence of gpx1 gene expression . in colon mucosa , the heterozygous double - ko has the same level of gpx activity as wildtype mice . the homozygous double - ko mice had a slower weight gain compared with mice of other genotypes starting around day 16 postnatally . the two double - ko mice had the same birth weight and maintained the same weight gain as their littermates until weaning . among the 33 double - ko mice followed , 32 showed growth retardation onset at 16 - 26 days old . the last one started to show growth retardation at 30 days . other symptoms often associated with these homozygous double - ko mice include perianal ulceration ( redness and irritation of anal region ), anal mucous discharge , and diarrhea . one or more of these symptoms occurred as early as 14 days old . however , most of these symptoms were transient except the perianal ulceration , which appeared to be persistent . older double - ko transgenic mice , over six months old , had a high level of tumor in the ileum . the younger homozygous double - ko mice had at least 25 % mortality . death or morbidity indicating imminent death occurred between 20 - 36 days of age . five of the 33 homozygous mice that we tracked daily died unexpectedly , three more of the 33 mice were terminated when they appeared moribund judging by persistent weight loss , hunched - over posture , or rectal obstruction . no noticeable abnormality was seen in major organs , for example such the liver , kidney , heart , lung , spleen or lymph nodes in the autopsy . in spite of the severe growth retardation , wasting syndrome , and mortality , the homozygous double - ko mice had similar weight and length of small and large intestine compared with their littermates up to 25 days old . after 40 days , the length and weight of small intestine in the homozygous double - ko mice began to lag behind their littermates by 20 %. however , the weight of colon and rectum in the homozygous double - ko mice was about 20 % heavier than that in their littermates . this may simply reflect the thickening of colon mucosa in the double - ko mice . to determine if the severe growth retardation was contributed by lack of calorie uptake despite of normal intestinal growth in the homozygous double - ko mice , we monitored the rectal temperature and amount of food uptake by these mice . we found these mice are hypothermic compared with their littermates either under normal housing condition or in metabolic cages where there was no bedding . referring now to fig3 a , there is shown a graphical representation of rectal temperatures of double - ko mice as compared to their littermates . rectal temperature of homozygous double - ko mice and their littermates with either combined heterozygous ko or three - quarter ko . double - ko mice are triangles and their littermates are squares . the error bars are variances or standard deviations from means of 2 - 6 mice . rectal temperatures of the younger ( 24 - 36 days old ) and more mature ( 40 - 67 days old ) double - ko mice were 37 . 0 ± 1 . 1 ° c . and 35 . 1 ± 2 . 2 ° c . respectively . the rectal temperatures of their littermates were 37 . 6 ± 0 . 6 ° c . for all ages under normal housing condition . after being placed in metabolic cages for 24 h , the rectal temperature of 36 day - old double - ko mice had dropped from 36 . 2 ± 2 . 3 ° c . to 32 . 2 ± 1 . 8 ° c . as shown in fig3 b . the control mice did not change their rectal temperature significantly after being housed in metabolic cages . the homozygous double - ko mice ( 24 - 49 days old ) consumed similar amounts of food ( 0 . 16 ± 0 . 07 g mouse chow / g body weight per day , n = 11 ) as their littermates ( 0 . 10 ± 0 . 05 g chow / g body weight per day , n = 18 ). the difference in food intake is not statistically significant . although the animals had bouts of acute diarrhea and loose stools , they did not have chronic diarrhea . referring now to fig3 b , there is shown a graphical representation of hypothermia caused by stress in double - ko mice . adult ( 36 - day - old ) mice were stressed by housing singly or doubly in metabolic cages for 24 hours . the error bars are variances of means from four double - ko mice and six littermates with gpx1 +/− gpx2 +/− and gpx1 −/− gpx2 +/− genotypes . histological analysis was performed on the cross sections of stomach , jejunum , ileum , colon and rectum after staining with hematoxylin and eosin as shown in fig4 . cross sections from two 20 day - old littermates with homozygous double - ko and 3 - quarter ko genotypes were compared . the 3 - quarter ko had apparent normal histology throughout the gi - tract . in contrast , the double - ko mouse had severe ileitis and colitis , although the jejunum and stomach appeared to be unaffected . crypt abscesses were prevalent in ileum , colon and rectum . the extent of ileitis and colitis were scored with the histological changes in five categories : ( 1 ) severity of the inflammatory cell infiltrate in lamina propia ; ( 2 ) epithelial cell reactive hyperplasia / atypia ; ( 3 ) mucin depletion ( colon and rectum only ); ( 4 ) increases in intraepithelial lymphocyte numbers in crypts ; and ( 5 ) number of inflammatory foci as defined previously ( 33 ). periodic acid shift ( pas ) staining was performed on some sections to confirm the depletion of mucin . referring now to fig4 there are shown the results of histology of mouse ileum , colon and rectum stained with eosin and hematoxylin . one 3 - quarter ko ( top row ) and one homozygous double - ko ( lower row ) littermates were sacrificed at 20 days of age . arrows point at crypt abscesses . the original magnification is 200 ×. table 1 shows the progression of ileitis and colitis from distal to proximal direction in 18 homozygous double - ko mice through early development . spontaneous colitis was shown mostly in the distal colon as early as 11 days of age in 6 out of seven mice analyzed . the proximal colitis was only observed in one of seven 11 - 14 days old mice analyzed . most mice of 15 days and older had inflammation in both distal and proximal colon . ileitis became evident and prevalent in mice of 20 - 27 days old . no inflammation was seen in the stomach and jejunum in all animals up to 60 days old ( table 1 and other observations ). other major organs including heart , liver , lung , kidney , testis , and brain did not have any noticeable abnormality upon gross and histological analysis . mice with disrupted single gpx1 and gpx2 genes are apparently normal . this raises some question as to the individual importance of each of these antioxidant enzymes . this lack of an observable deleterious phenotype in single knockout mice also suggests that animals have overlapping defense system against hydroperoxides , since catalase , glutathione s - transferases and aop - 2 can reduce some species of gpx substrates ( 34 - 37 ). in contrast to the single knockout mice , the gross abnormality found in mice with combined disruption of gpxl and gpx2 genes demonstrates the uniqueness of gpx activity &# 39 ; which cannot be compensated by other types of hydroperoxide - reducing enzymes . this result also suggests that gpx - 1 and gpx - gi are functionally redundant . the gpx - 1 appears compensating for lack of gpx - gi in epithelium of small intestine judged by the same level of gpx activity detected in mice expressing 0 and 1 gpx2 allele . a higher level of gpx - 1 in homozygous gpx2 - ko intestine was detected compared to the gpx - 1 level in wildtype mice determined by immunoprecipitation ( 4 , 13 ). the same level of gpx - gi was detected in gpx1 - ko intestinal mucosa . these observations suggest the gpx1 gene compensates for lack of gpx2 gene expression , but not vice versa . the compensation appears to be limited to small intestine but not colon . alternatively , it is also possible that a part of the expression machinery necessary for selenoproteins in favor of gpx1 but not gpx2 gene expression is active in the intestine but not in colon epithelium . this selenoprotein expression machinery includes 3 ′- untranslated region selenocysteine insertion sequence ( secis ) in mrna ( 38 ), selenocysteine trna [ ser ] sec ( 39 ), a secis binding protein named sbp2 ( 40 , 41 ), and mammalian upfl protein ( also known as rent or regulator of nonsense transcripts ) ( 42 ), etc . it is not clear if any of these factors differentiate between gpx1 and gpx2 mrnas . the same gpx level in colon mucosa of wildtype control and heterozygous double - ko mice suggests that this expression machinery for selenoproteins may be a limiting factor . it is clear that the double - ko mice have almost no gpx activity in the mucosa of distal gi - tract . although 3 - quarter ko mice with no gpx1 alleles have only a small fraction of total gpx activity in the distal gi - tract , this low level of activity appears to sufficient to maintain normal physiology . in fact , rodent gi - epithelium may have one - fold higher gpx activity compared with that in humans . the specific activity of gpx in human intestine and colon mucosa is 100 - 240 mu / mg protein compared with 300 - 700 mu / mg in rats ( 4 ) and mice . although the difference in gpx activity level in the gi - tract is not as big as that in liver , where humans have 352 + 89 mu / mg ( 43 ) and rodents have ˜ 4 , 000 mu / mg ( 44 , 45 ), the lower gpx activity level in human gi - tract suggests its higher susceptibility to peroxidative injury . the first sign of abnormality observed in these double - ko mice is growth retardation . it is well documented that severe se - deficiency causes growth retardation in young animals ( 46 ). injection of triiodothyronine ( t 3 ) to restore plasma thyroid levels in these se - deficient animals did not increase animal weight gain ( 47 ). since gpxs are se - dependent enzymes , this slow growth caused by se - deficiency in 2 nd generation rodents can be explained by lack of gpx - 1 and gpx - gi in the gi - tract . this suggests that these 2 nd generation se - deficient animals should be examined for colitis . to determine if growth retardation in the homozygous double - ko mice is due to lack of food intake , mice were placed in metabolic cages to monitor the amount of food , water and excretion for a 24 - hour period . often , two mice were placed in one metabolic cage since the double - ko mice could not sustain the stress well when housed alone in this setting . the stress may be contributed by the cooler air due to lack of bedding and shelter . since the double - ko mice consume the same amount of food as their littermates , and do not have chronic osmotic diarrhea , it is possible that these double - ko mice are either deficient in converting the calorie intake into metabolic fuel as implicated in the older gpx1 - ko mice ( 12 ), or suffering from inflammation - induced cachexia ( 48 ). many mammals respond to energy deficit , such as calorie restriction , by lowering body temperature ( 49 ). in fact , fasting can induce torpor or extreme hypothermia in mice ( 50 ). since these mice have wasting syndrome , we wanted to determine if they also have hypothermia consistent with deprivation in metabolic energy . the hypothermia presented in these mice support the notion that these mice may not be getting enough calories despite unrestricted access to food and normal appetite . it will readily be appreciated by those skilled in the art that determination of hypothermia in mice of the present invention supplied with a high fat diet can be utilized to answer this question . animals suffering from inflammatory bowel disease ( ibd ) often show wasting ( 33 ). the double - ko mice tend to have colon inflammation , which shows up as a thickened colon and heavier colon weight ( 24 ). histological study shows that these homozygous double - ko mice have spontaneous inflammation starting from distal colon as early as 11 days old , which was the youngest age analyzed . inflammation progresses from distal colon to proximal colon and then to ileum . the increased severity in colonic inflammation around weaning appears to be correlated with the increased number of species of colonic bacteria ( 25 ). the alteration in colonic bacterial flora can result from either ingestion of solid food which alters luminal ph ( 51 ) or decreased in the protective iga and other bactericidal components present in milk ( 52 ). the severity and timing of ileitis and colitis in these double - ko mice is consistent with the notion that microflora is an important cofactor in the pathogenesis of colonic inflammation ( 24 , 25 ). germ free mice of the present invention can be utilized to make this analysis . reactive oxygen species have been implicated in the pathogenesis of ibd . the inflamed colon has elevated levels of oxygen metabolites detected by chemiluminescence ( 27 , 28 ). catalase , superoxide dismutase , or azide ( a myoleperoxidase inhibitor ) decreases chemiluminescence . compounds used for ibd therapy such as 5 - aniinosalicylates have antioxidant activity ( 26 , 53 ). thus , increased oxidative stress may play an important role in the pathogenesis of ibd . selenium deficiency is common in those patients with severe gastrointestinal disorders due to impaired intestinal absorption ( 31 , 32 ). however , little evidence supports the role of gpx in pathogenesis of ibd . thus , the ibd phenotypes presented in these double - ko mice provide the first evidence to link gpx activity with this disease affecting one million americans . it will be appreciated that the methods and compositions of the instant invention can be incorporated in the form of a variety of embodiments , only a few of which are disclosed herein . it will be apparent to the artisan that other embodiments exist and do not depart from the spirit of the invention . thus , the described embodiments are illustrative and should not be construed as restrictive . 1 . ursini , f ., heim , s ., kiess , m ., maiorino , m ., roveri , a ., wissing , j . & amp ; flohe , l . 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