Strain restricted typing sera (SRTS) were produced for some of the most used inbred mouse and rat strains in Denmark by injecting lymphocytes pooled from several different strains intraperitoneally into the recipients. The SRTS were combined with a complement dependent cytotoxieity assay. It was possible to distinguish animals from strains with different MHC haplotypes but not animals from strains with the same MHC haplotype. It was concluded that the SRTS eomhined with a&nbsp; complement dependent cytotoxicity assay could distinguish between some of the most commonly used inbred strains of rats and mice in Denmark

Dagnaes-Hansen, Frederik

Hansen, Axel Kornerup

Jensen, Lene

English

Journals from University of Tartu

Scand.J.Lab.An:m.Sc1. V0.3 . 1992\101. 1‘)Strain restricted typing sera for the use in the geneticmonitoring of inbred strains of mice and ratsfrom two danish SPF breedersby Lene .lemen', AerKamerup Hansen2 & Frederik Dagmes-Il'anspn31 Department of Animal Science and Health, Division of Animal Genetics, The Royal Veterinary andAgricultural University. Copenhagen, Denmark, 1 Mellegaard Breeding Centre Ltd, Ll. Skensved,Denmark, 3 Bomholtgfird Breeding and Research Centre Ltd, Ry, Denmark.IntroductionEven under optimal conditions in breedingfacilities, it is necessary to control the inbredstrains of laboratory rodents for geneticauthenticity, as contamination of breedingstocks have been known to occur (Krog1976, Cramer & Silver 1981, Fasting 1982,Penline er a1. 1982). Ideally the genetic mo-nitoring method of choice should be fast,convenient, inexpensive and accurate (Cra-mer 1983).Strain Restricted Typing Sera (SRTS) areantisera which are produced by immunizingone strain with lymphocytes from strainsrepresenting all other MHC-types in thesame unit. The SRTS are described in assayscombined with a complement dependentcytotoxicity test (Fasting & Talmcm 1980,Am et a]. 1982, Kendall & Wagner 1985)and in an assay with hemagglutination (Ken-dall & Wagner 1985). Ideally the reci-pient strain should react toward all otherstrains in the unit.Some of the advantages offered by the assayusing SRTS are that the test is simple, reli—able and repeatable (Am et a1. 1982). It is aninexpensive method and with some trainingone technician can monitor about 10 ani-mals per day. Using this assay it is primarilythe MHC haplotype which is monitored.This is an advantage, as the haplotype isimportant in many scientific experimentswhere laboratory rodents are used (Gi‘inther1990). Further, it is an advantage that theantisera can be produced easily in the labo-ratory using the inbred strains which arebred on site (Fasting & Totman 1980, Ken-dall & Wagner 1985). However, it must bepointed out that rodents used to produceSRTS must be genetically authentic for theSRTS produced to give valid results (Kerrdull & Wagner 1985).The aim of this paper are to describe theproduction of SRTS for some of the mostused inbred strains of mice and rats in Den-mark, and to investigate the reaction patternbetween strains having the same or differentMHC haplotypes.Materials and methodsAnimalsFor the production of the strain restrictedtyping sera (SRTS) animals from seven in-bred strains of mice and five inbred strainsof rats were used as recipients (sera produ-cer). At the beginning of the experiment theage of the animals was about six weeks. Aslymphocyte (spleen) donors for immuniza-tion, animals from seven inbred strains ofmice and 16 inbred strains of rats were used(see Table 1 & Table 2). Age and sex werenot kept constant for the donor animals.The strains were from the suppliers Bom-holtgéird Breeding and Research Centre Ltd,(Ry, Denmark) and Mallegaard BreedingCentre Ltd. (L1. Skensved, Denmark), wherethey were bred under barrier conditions.Antisera productionThe SRTS were produced as described byFasting & Tolman (1980). Spleens from thedonors Were removed and a single cell sus-97Scand. J. Lab. Anim. Sci. No. 3. 1992 .V01. 19Table l. Mice used as recipients and donors forproduction of SRTS. Table 2. Rats used as recipients and donors forproduction of SRTS.  Strain Sex Strain SexRecipients RecipientsA/J Bom M BB/Wor/Mol—BB FBALB/CJ Born F BB/Wor/Mol-WB FC57BL/6J Bom F LEW/Mol FCBA/J Bom M PVG/Mol FNOD M SHR/BM FNZW/X Born 1“ .Donor strainsSJUN 30‘“ M BB/Wor/MQLBBDonor strains BB/Wor/Mol—WBA/J Bom BN—MolBALB/cJ Rom BUF/MolC57BL/6J Bom Dahl—R/MolCBA/J Born Dahl—S/Mol.\0D LE/MolXZW/N Born LEWI'MnlSJL/N Bom NEDH/MolPVG/MolSHR/‘BMSHR/‘Molpension was prepared in PBS (phosphatebuffered saline) by cutting the spleens fol-lowed by filtration through gauze. Splenielymphocytes were pooled from all the donorstrains and injected intraperitoneally (i.p.) inthe recipient animals.The recipient groups consisted of seven miceor five rats of each strain. For each immuni-zation, the recipients were injeted withpooled lymphocytes from the donor strains.For each rat immunization one spleen fromall the donor strains was used per ten reci-pients, whereas for the mice, one spleenfrom all ofthe donor strains was used for allthe recipient (altogether 49 mice).The recipients received an injection ofpooled lymphocytes once a week for fiveweeks. A week after the last injection, theanimal were bled, the serum pooled withineach strain and frozen at ~20“C.Preparation oftest lymphocytesuspensionThe animals were killed by cervical disloca-tion or in a carbon dioxide gas chamber.One or more lymph nodes were removedand placed in approximately 1 ml of cellmedia (RPMI 1640, hepes buffered (Sigma,St. Louis, USA), with 1% glutamine and98Sprd/M01 (Inbred)Stroke—Prone/MolWF/MolVVKY/Mol 0,3 0/0 bovine serum albumin or 10% fetalcalf serum). The lymph nodes were teasedapart with fine scissors and the cell suspen-sion filtrated through cotton wool. Thelymphocytes were washed twice in cellmedia, and then the cell suspension wasadjusted to 2—3x106 cells per m1.Complement dependent cytotoxicityassayTwo different types of assays were used inthe cytotoxicity test. The one performed inU bottom microtiter plates and the otherperformed in Terasaki plates. In each assayall animals were tested with all the SRTS forthe species tested. In each assay all animalswere tested with all the SRTS for the speciestested. In each assay. there was a positiveand a negative control for the SRTS and forthe complement (guinea pig serum).Assay performed in microtiter plates.The assay was with some modifications per—formed as previously described (Nielsen &110131 1983, Gilnlhcr 1990). Twenty ul ofantisera diluted in PBS was pipetted intoeach well. Twenty ttl of lymphocyte suspen—sion was added, the suspension [nixed wellwith a multipipette. then the microtiter platewas covered with tinfoil to avoid evapora—tion and incubated at 37C for 30 min. Fifty11.1 of undiluted guinea pig serum was added,the suspension mixed well with a multi-pipette and then the microtiter plate wasincubated at 37°C for 60 min. Twentyfive 1110f0. 5 0/o trypanblue (Sigma St. Louis, USA).in O9 0/11 saline was added to about 10 wellsat a time and mixed carefully with a pipette.After a couple of minutes the supernatantwas carefully sucked up with a pasteurpipette to leave the cells laying on the bot—SeandJ, Lab,An1m. Sci. No 3,1992.V01.19tom of the wells. Immediately after the re-moval of the supernatant, the proportion ofdead cells (blue) among live cells (colourless)was determined by estimation, using an in—verted microscope with 100 x magnification.Assay performed in Terasaki plates.The assay in Terasaki plates (Life Tech-nologies A/S, Roskilde, Denmark) was per-formed as described (Terasaki 1964, Festing& Totman 1980, Giinther 1990) with somemodifications dependent on the laboratoryfacilities. Two 111 of antisera diluted in PBSwas pipetted into each well. Two 111 of lym—phocyte suspension was added, the suspen-sion was mixed by carefully shaking theplate and incubated at 37“C for 30 min. ToTable 3 Results of testing seven mouse strain restricted typing sera against lymphocytes trom sevenstra1ns. Major histocompatibility haplotypc in brackets. Obtained by testing in microtiter and Terasakiplates. The degree of cytotoxic activity 1s proportional to the pluses where O is no cytotoxic activity and+H- is maximal eytotoxie activity. Thequotation beneath the degree of eytotixic activity indicates the tmaldilution giving results. Strains in which SRTS were producedLymphocyte A/J BALB/c] C57BL/6J CBA/J NOD NZW/N SJL/Ndonor (:1) (d) (b) (k) (g) (I) (S)/J 0 +1+ +1+ +1+ 1+1» +H-(a) 1:4 1:4 1:4 1:8 132 1:4 1:16BALB/‘CJ +1--+1+ 0 +1+ -H-1- +1+ +1+ +1+((1) 1:4 1:4 1:16 1'8 1:32 1:8 1:16C57BL/6J +1—+1+ +1+ 0 +++ +1+ +1+ +1+(b) 1:4 1:4 1:4 18 1:32 1:4 116CBA/J +-+1- +H- +1+ 0 +H- +1 +1+(k) 1:4 1:8 1:4 1:4 1:32 1:4 1 8NOD H—H—l— ++ +++- H—4—H 0 +1+ +H-(g) 1:4 1:4 1:4 1:8 1:4 1:4 1:8VZW/N H -H+ -H-1- +1+ +14 0 +1+(7) 1:4 1:4 1:8 1:4 132 1:4 1:16SJL/N +1—+—++ 4+ 4+ +1+ +1+ -1-H- 0('5) 1:4 1:4 1:4 1:8 1:32 1:8 1:4BALB/CA* 4+ 0 +1+ ++ +H- +1+ +1+(d) 1:4 1:4 1:4 18 1:32 1:4 1:8C311/’11e]* 0—+ +1+ ++--H—1- 0 +1+ + +1+(k) 1:4 1:4 1: 14 1:32 14 1:8C57BL/ +1+ +1+ 0 4+1 +1+ +1+ +H-103c1'*(b) 1:4 1:8 1:4 18 1:32 14 l 16DBA/21* +H- 0 +++ +1+ +H- +1+ +1+(d) 1:4 1:4 1:8 1 16 1:32 18 1 16* Only 1—2 animals per strain tested. The recipient animals are not immunized against these strains.99Seand.J.Lah.An1m.Sci No 3 1993.1/(11 19avoid evaporation from the wells. water wasplaced in the bottom of the plate. Then 5 111of undiluted guinea pig serum was added toeach well, the plate was carefully shakenand incubated for 60 min. Two 111 of 7%Eosin Y (Sigma, St. Louis, USA). was thenadded and the plate shaken. Within half anhour, the reading of the assays began, usingan inverted microscope with phase contrastat 100 x magnification. In each well. theproportion of dead cells (dark) among livecells (bright and smaller) was determined byestimation.Evaluation ofthe results.In the microtiter plates the negative controlwas accepted when the proportion of deadcells was less than 15 0/0. 1n the Terasakiplates, 20~25 % dead cells were accepted.The degree of cytotoxic activity was evalua-ted as 0 : cell death equivalent to the celldeath observed 111 the negative control, + =40—60 0/0 cell death, -1+ = 60—80 % cell deathand +1+ = 80—100%ee11 death.ResultsMost of the results given in Table 3 andTable 4 were tested in microtiter plates. asthere were some technical problems with theuse of the Terasaki plates. 1n the Terasakiplates, negative control for SRTS was15—50 0/0 dead cells. where it generally wasless than 10% 111 the microtiter plates. Thetests performed in Terasaki plates, which re-acted satisfactory. gave a cell death similarto the cell death in microtiter plates aboutthe same dilution of antisera.The results presented in Table 3 and Table4 were obtained by testing at least two andusually four animals from each strain witheach ofthe SRTS for the species.As seen in Table 3. within the seven strainsof mice used for the immunization (all withdifferent H~2 haplotype), the serum testedagainst target cells from the same strain gavea negative result in all cases. as predicted.Among the four extra strains tested whichwere not used in the immunizing of the re-cipient animals, it can be seen that exceptTable 4. Results of testing five strain restricted typing sera against lymphocytes from fivestrains obtained by testing in mierotiter and Tet‘asaki plates. Major histocompatibilityhaplotype in brackets. The degree of cytotoxic activity is proportional to the pluses, where 0is no cytotoxic activity and +1+ is maximal cytotoxic activity. The quotation beneath thedegree ot‘cytotoxic activity indicates the final dilution giving results. Strains in which SRTS were producedLymphocyte BB/BB BB/WB LEW PVC SHR/BMdonors (u) (u) (1) (C) (k)BB/BB O 0 +H- ++ +1+ +1+(11) 1:4 1:4 1:32 1:8 1 8BB/WB O 0 +1+ +1- 4+(u) 1:4 1:4 1:32 1:4 1 8LEW +1» +++ 0 +H- +H-(1) 14 1:16 1:4 1:16 1:16PVG +1+ +1+ +H- 0 +1+(c) 1:8 1:32 1:64 1:4 1 16SHR/BM +1+ +1+ +1+ +1+ 0(k) 1:4 1:32 1:64 1:16 1:4LE* 0 0 +1+ 4+ -H—-1+1-(11) 1:4 1:32 1:32 1 8 1:16 * Only one animal of the strain tested.100for C3H/HeJ, the SRTS reacted with lym-phocytes from strains with another H—2haplotype than the haplotype from the im-munized strain.From Table 4, it can be seen that the samepattern of reaction occur within the ratstrains as for the mice. The serum testedagainst target cells from the same strain gavea negative result, and serum tested againsttarget cells from a strain with the samehaplotype as the test serum also gave a nega-tive result. Another interesting thing inTable 4 is that the c MHC-haplotype (PVG)especially but also the k MI-IC—haplotype(SHR/BM) had difficulties in reacting withthe u MHC-haplotype, but that there wereno problems the other way around as theBB/WB is one of the best antisera producersamong the five strains immunized.As seen in Table 3 and Table 4, the seraconcentration needed depended on whichstrain the antisera were produced in andwhich strain was tested. It is seen thatamong the mouse strains, A/J, BALB/e] andNZW/N were not too good in producingSRTS, whereas NOD was much better thanthe other strains. Among the rats BB/BB wasa poor producer of SRTS, which was expec-ted as the strain is immunodeficient. Espe-cially LEW but also BB/WB were good inprod ueing SRTS.DiscussionIn the experiments reported in this paper, itwas not possible to distinguish strains whichhave the same MHC-haplotype, while Fest-t'ng (1’1 Tolman (1980) and Kendall & Wag—ner (1985) could partly distinguish withinthe same haplotype. If a genetic contamina-tion was detected it would not have beenpossible to find the source of contamination,as the test lymphocytes would have been k11-led by all the test sera if the contaminanthad been a Fl-hybrid.Two different assays were used for the cyto—toxicity test. The one carried out in micro-titer plates gave the most stable results, butwas more Iaborious than the assay carriedScand. J. Lab. Anim. Sci. No. 3 . 1992 . Vol. 19out in the Terasaki plates. Moreover, theassay carried out in the Terasaki plates didnot use so much SRTS and other reagents,and it was faster to carry out. But the assaydid not work optimally, the backgrounddeath of lymphocytes being 15—50 0/11, whichis unacceptably high. One reason could bethat the quantities when using Terasakiplates are very low, resulting in either lackof nutrients, pH changes or a general toxi—city of one of the reagents.The reason, why our assay was less succesfulwhen performed 111 Terasaki plates might bedue to the fact that the trays were incubatedat 37" rather than at room temperature. Thismay account for the high background rate ofcell death. Preliminary filling of the wellswith paraffin oil might also have improvedthe results.Reactions of e.g. the mouse strains A/J andNZW and the rat strain BB/BB are weak.These results might have been improved bytesting with undiluted serum as done byFevlmg er a]. (1980).In the assay carried out in the Terasaki pla-tes, it seems as if lymphocytes from somestrains were less capable of surviving thanlymphocytes from other strains (e.g.C57BL/6J. C57BL/IOSe and NOD amongthe mouse strains, and BB/BB and BB/WBamong the rat strain did not survive well).The antisera concentration needed wasabout the same in the two methods.SRTS produced in A/J and NZW/N reactedbadly with Iymphocytes from C3H/HeJ, butthis could possibly be explained by thesetwo sera being weak.When comparing the concentration of SRTSneeded for the eytotoxieity test, it was seenthat the rat SRTS were more concentratedthan the mouse SRTS. This could be be-cause the rats were immunized with approx-imately five times as many lymphocytes asthe mice.In the experiments hei ; reported. it was pos-sible to distinguish strains with ditIerenthaplotype by means of SRTS combined witha complement dependent cytotoxicity assay[0/Stand] Lab.Anim.Sei.No.3.1992.Vol.19primarily carried out in microtiter plates.but it was not possible to distinguish strainswithin the same haplotype. This means thatthe use of SRTS combined with a cytotoxi-city assay is a valuable tool in the geneticmonitoring in combination with other me—thods e:g. other serologieal and biohemicalmarkers.AcknowledgementsWe thank lecturer Knud Christensen from De—partment of Animal Science and Health, Divisionof Animal Genetics, the Royal Veterinary andAgricultural University, Copenhagen for readingand commenting the manuscript.SummaryStrain restricted typing sera (SRTS) were pro-duced for some of the most used inbred mouseand rat strains in Denmark by injecting lympho—cytes pooled from several different strains intra-peritoneally into the recipients. The SRTS werecombined with a complement dependent cytotoxi-eity assay. It was possible to distinguish animalsfrom strains with different MHC haplotypes butnot animals from strains with the same MHChaplotype. It was concluded that the SRTS eom-hined with a complement dependent cytotoxicityassay could distinguish between some of the mostcommonly used inbred strains of rats and mice inDenmark.SammendragStamme begraensct type sera (SRTS) blev produoe~ret for nogle a1 de mest brugte indavlede muse- 0grottc-stammer i Danmark, ved at injicere lymtb-eytter blandet fi'a forskellige stammer intraperito-nealt 1 recipienteme. SRTS’et blev brugt 1 enkomplement afheengig eytotoxicitetes test. Det varher muligt at adskille dyr t'ra stammer med for-skellige MHC haplotyper, men 1kke dyr fra stam-nier med den samme MHC haplotype. Det blev102konkludcrct, at SRTS kombineret med en kom-plement alhaengig eytotoxieitets test 1(an skelnemellern nogle af de mest brugte rotte— og muse~indavlsstammer 1 Danmark,ReferencesAm JS, SE Riordan, D Pearson & DI] Sar‘hs.‘Strain restricted typing sera (SRTS) for use inmonitoring the genetic integrity of eongeniestrains. 1. 1mm. Met. 1982, 55, 141—153.Cramer DW & WK Silver: Workshop report.Genetic uniformity in inbred rat strains.transpl. proc. 1981,13, 1492—1493.Cramer DV: Genetic monitorings techniques inrats. ILAR News 1983, XXVI, 15—19.Fasting MFW & P I'otman.’ Polyvalcnt strain—spccifie alloantisera as tools for routine gene-tic control of inbred and eongenie strains ofrats and mice. Lab. Anim. 1980, 14, 173—177.Festmg MFW: Genetic contamination of labo—ratory animal colonies: An increasingly seriousproblem. ILAR News 1982, 25, 6—10.Giim‘her E: Immunological markers. In ”Geneticmonitoring of inbred strains of rats”, H] Hed-rich, 1311., Gustav Fischer Verlag, Stuttgart,New York 1990.Kendall (7 & J}? Wagner: Characterization ofstrain specific typing antisera for genetic mo—n1toring of inbred strains of rats. Lab. Anim.Sci: 1985, 35, 364—369.Krog HH: Identification of inbred strains of mice,Mus .Musculur. 1. Genetic control of inbredstrains of mice using starch gel-eleetrophore-sis. Bioehem. Genet. 1976, 14, 319—326.Nielsen 1M & H Holst: Cytotoxicity test, usingmonoclonal H—Z antibodies in the genetic con—trol program for mice. GV—SOLAS XXI meet—ing, Miinster 1983.Pennline KP, JP Smith & H Biller-Suermann:My kingdom for an inbred rat. Transplanta-tion 1982, 34, 70.Teramki PI & JD MrCle/[amls Mierodmplet as-say of human serum eytotoxins. Nature 1964,204, 998—1000.

Strain restricted typing sera for the use in the genetic monitoring of inbred strains of mice and rats from two danish SPF breeders

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Strain restricted typing sera for the use in the genetic monitoring of inbred strains of mice and rats from two danish SPF breeders

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