ILAR Journal Online, Volume 33(3) 1991

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ILAR Journal V33(3) 1991

RT1A (MHC), RT2, and RT3 (Blood Group) Specificities of 44 Inbred and Congenic Rat Strains from the NIH Genetic Resource
Heinz W. Kunz, Ph.D., Barbara Dixon-McCarthy, Maria A. Lepre, Carl T. Hansen, Ph.D., and Thomas J. Gill III, M.D.

This report was prepared at the University of Pittsburgh School of Medicine, Department of Pathology, Pittsburgh, Pennsylvania, and at the National Institutes of Health, National Center for Research Resources, Bethesda, Maryland. At the University of Pittsburgh, Heinz W. Kunz, Ph.D. is professor of pathology, Thomas J. Gill III, M.D., is Maud L. Menten professor of experimental pathology and professor of human genetics, Barbara Dixon-McCarthy is a research specialist IV, and Maria A. Lepre is a research specialist Ilk Carl T. Hansen, Ph.D., is a geneticist at the Genetic Resource Unit, National Center for Research Resources, National Insitutes of Health.

INTRODUCTION

The laboratory rat (Rattus norvegicus) is extensively used in experimental transplantation, cancer research, immunology, and toxicology. There are well over 100 different strains of rats listed in Inbred Strains in Biomedical Research (Festing, 1979); however, very few of these strains are used regularly by investigators worldwide. Many of these inbred strains with the same name were independently derived from outbred stock; therefore, these strains may vary genetically. It is desirable that these strains be genetically uniform and constant, and establishing genetically defined reference strains of rats would help to ensure uniformity. Regular monitoring of the animals' genetic profiles is also necessary to ensure that these strains remain genetically homogeneous. Genetic monitoring is customarily accomplished by testing each strain for a set of biochemical markers, such as polymorphic proteins and enzymes, and immunologic markers. Recently, molecular techniques have also been employed.

Immunologic markers, such as histocompatibility antigens (MHC) and blood group antigens, are extremely important parameters in experimental transplantation, and they can be employed as useful markers for genetic monitoring. These antigens can be determined with relative ease, and a large number of well-defined reagents for determining them are available.

For many years, rats from the NIH Genetic Resource have been widely used as models in transplantation research. However, many of these strains have never been tested for their histocompatibility type and/or blood group antigens. Here we report RT1.A (MHC class I antigens), RT2, and RT3 (blood group) specificities of 44 inbred and congenic strains of rats from the NIH Genetic Resource.

MATERIAL AND METHODS

Table 1 lists the monoclonal and polyclonal antibodies that were used for determining the RTl.A, RT2, and RT3 specificities. Their immunoglobulin isotypes, the strain combinations in which the antibodies were raised, and their respective specificities are also shown. Production of monoclonal antibodies has been described previously (Misra et al., 1981). Several examples will illustrate this approach. Monoclonal antibody 211-4-D9, which was produced by immunizing the (MR x WKA)F₁ hybrid strain (RTl .A^o/k) with lymphocytes from the DA (RTl .A^a) strain, is specific for the A^a antigen and will react only with all strains of rats carrying the RT1.A^aspecificity. Monoclonal antibody 3-5-118 will react with several different haplotypes and serves as a useful reagent for the general detection of class I MHC antigens.

Affinity-purified monoclonal antibodies to MHC class I specificities were biotinylated, and their binding to peripheral blood lymphocytes was measured with FITC-conjugated avidin using an Epics Profile II flow cytometer. Some monoclonal antibodies were used in concentrated and purified form, and their binding was measured with FITC conjugated to rabbit anti-rat IgG (Fab')2. Figures 1 and 2 show flow cytometric analyses of the monoclonal antibodies to the RTl.A specificities with the appropriate negative and positive controls. Measurement of fluorescence intensity (degree of binding) is shown on a log scale. Positive reactions show 90% or greater binding of fluorescein isothiocyanate (FITC)-conjugated antibody to lymphocytes and negative reactions show less than 5% binding.

Hemagglutination in Ficoll (Kunz and Gill, 1974) was used to assay for RTl.A^g with polyclonal antibody No. 7548. This method was also used to assay for RT2 and RT3 specificities using both polyclonal and monoclonal antibodies.

RESULTS

The reactivity patterns of 44 inbred and congenic strains of rats from the NIH Genetic Resource with the panel of monoclonal and polyclonal antibodies are shown in Table 2. Two animals (siblings) of each strain from pedigreed parents were tested, and their generation of inbreeding is listed. The RT1.A haplotype, as deduced from the reactivity pattern, and the RT2 and RT3 (blood group antigens) specificities are given.

There were no equivocal reactivities detected. The reactivity pattern and deduced RTl .A^c haplotype for the MNR/ AN strain was unexpected, because the prototypic MNR strain had been described by Stark et al. (1978) as having the RTl^m (formerly H-1^m) haplotype and as showing reactivity patterns with antibodies to class I antigens similar to those of the MR/N strain but having the RT1B/D^c (class II) specificity. Thus, the MNR strain is a natural recombinant strain; it was later designated as the prototypic strain of the m haplotype (RT1.A^dB/D^c). Some MNR strains of British origin had been typed earlier by Kren (1974).

There have been reports that some BUF strains have the RT1^a haplotype and that the RT2^b specificity has been found in some colonies of BN rats. The results from this study show that the BUF/N at F134 has maintained the RTl^bhaplotype, as described originally by Palm (1971) and by Stark et al. (1979), thus confirming the strain's genetic purity. It should be used as the prototypic strain for the RTl^b haplotype. Similarly, the BN/SsN strain at F86 still types for the RT2^a and RT3^bspecificities as described earlier in the BN strain obtained from Dr. Carl Hansen in 1974 (Kunz and Gill, 1978). In summary, MHC specificities and blood group antigens serve as useful markers for genetic monitoring of inbred rat strains and should be used in addition to biochemical markers and restriction fragment length polymorphism (RFLP) mapping. In view of the extensive use of rats in experimental transplantation, knowledge of their transplantation antigens is critical.

REFERENCES

Festing, F.W. 1979. Inbred Strains in Biomedical Research. New York: Oxford University Press.

Kren, V. 1974. The major histocompatibility system (H-I) alleles of some British rat strains. Transplantation· 17:148-152.

Kunz, H. W., and T. J. Gill Ill. 1974. Genetic studies in inbred rats. I. Two new histocompatibility alleles. J. Immunogenet. 1:413-420.

Kunz, H. W., and T. J. Gill III. 1978. Red blood cell alloantigenic systems in the rat. J. Immunogenet. 5:365-382.

Misra, D. N., S. A. Noeman, H. W. Kunz, and T. J. Gill III. 1981 Production of monoclonal antibodies to rat MHC antigens using different myelomas. Transplant. Proc. 13:1347-1355.

Palm, J. 1971. Immunogenetic analysis of Ag-B histocompatibility antigens in rats. Transplantation. 11:175-183.

Stark, O., H. W. Kunz, and T. J. Gill III. 1978. Comparison of the haplotypes of the major histocompatibility complex in the rat. III. Two difficult haplotypes: H-I^h(Ag-BI2) in the HW strain and Ag-B13 (H-I^m) in the MNR/N strain. J. Immunogenet. 5:261-273.

Stark, O., H. W. Kunz, and T. J. Gill III. 1979. Comparison of the haplotypes of the major histocompatibility complex in the rat. IV. The six original Ag-B haplotypes. J. Immunogenet. 6:115-127.

TABLE 1 Monoclonal and Polyclonal Antibodies Used for Typing Inbred and Congenic Strains

Antibody no.	Immunoglobulin isotype	MHC Strain combination	RTI.A specificity
211-4-D9	G2b	(MR x WKA) anti-DA	a
152-2-H10	m	(MR x WF) anti-DA	a, k
3-5-118	G2b	WF anti-Da	a, b, d, f, m
402-1B4	m	(WF x DA) anti-BUF	b
426-2E9	G2b	(DA x BN) anti-AUG	c
303-C l-G2	G2b	(DA. 1F x WKA) anti-DA. 10(MR)	d
336-1B 1 I	G2b	(WKA x DA. 10) anti-DA. 1F(AS2)	f
396-C5	m	(DA x WF) anti-WKA	k
163-7F3	G2b	(DA x BN) anti-BN. IL (LEW)	l
42-1-17	G2a	[BN.1U(WF) x BN.1A(DA)] anti-BI	n
506-D4	¹	(WF x ACP) anti-BN	n, c
68-3-D1	Gl	DA anti-YO	u
70-3-C2	G2b	DA x BN) anti-DA. II(BI)	u, c²
381-1D-10	G2b	WF x DA pregnancy serum	Pa(a,b,d,f,m)
Polyclonal 7548		DA x BN) anti-KGH	g,b,c,u 1 Isotype unknown

¹ Isotype unknown
² The mAb with RTI.E^uspecificity also reacts with RT1.A^uand RTl^c

		NON-MHC
Antibody no.	Immunoglobulin isotype	Strain combination	Specificity
Polyclonal 5945		OKA anti-WKA	RT2^a
62-1-A3	G2a	F344 anti-NBR	RT2^b
Polyclonal 6659		F344 anti-LEW	RT3^a

Figure 1 Binding of various monoclonal antibodies to peripheral blood lymphocytes and the appropriate negative control.

Figure 2 Binding of various monoclonal antibodies to peripheral blood lymphocytes and the appropriate negative control.