Identification and evaluation of major histocompatibility complex antigens in chicken chimeras and their relationship to germline transmission

BREEDING AND GENETICS Identification and Evaluation of Major Histocompatibility Complex Antigens in Chicken Chimeras and their Relationship to Germline Transmission L. D. Bacon,*,1 L. Zajchowski,†,2 M. E. Clark,† and R. J. Etches†,3 *USDA, Agricultural Research Service, Avian Disease and Oncology Laboratory, 3606 East Mount Hope Road, East Lansing, Michigan 48823; and †Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada, N1G 2W1 unique to the WL line, and B*13-like and B-15-like genes were unique to the BR line, whereas a B*21-like gene was present in both lines. In adult BR→WL chimeras, as well as 10- to 14 d-old WL→WL chimeras, donor-type B antigens were detectable and quantifiable on RBC using flow cytometry. In BR→WL chimeras, the percentage germline transmission was significantly correlated with the percentage of RBC with donor B antigen, as well as percentage of black feathers in the plumage. In a retrospective study using previously developed BR→WL chimeras, the level of chimerism and germline transmission was higher in B*21/*21 type recipients, but this was not statistically significant in two prospective studies. It was concluded that MHC antigens on RBC can be used for identifying, quantifying, and selecting chicken chimeras developed by the transfer of blastodermal cells.

(Key words: major histocompatibility complex, B haplotype, chimera, germline, transgenic chicken) 2002 Poultry Science 81:1427–1438

INTRODUCTION Transgenic technology has been used to insert genes into the germlines of mice and other mammalian species to study the expression and function of genes. The development of transgenic chickens is desirable for similar reasons. However, the principle method used for mammals, i.e., microinjection of genetic material into a male sperm pronucleus prior to fertilization of an ovum (Palmiter and Brinster, 1985), has not been adaptable to chickens due to differences in the reproductive systems (Sang and Perry, 1989). An alternative method in mice uses transfected embryonic stem (ES) cells that are transferred into recipient blastocysts to produce chimeras that are

2002 Poultry Science Association, Inc. Received for publication December 18, 2001. Accepted for publication April 9, 2002. 1 To whom correspondence should be addressed: [email protected]. 2 Current address: Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N4N1, Canada. 3 Current address: Origen Therapeutics, 1450 Rollins Road, Burlingame, CA 94010.

bred to produce transgenic progeny (Gosler et al., 1986; Robertson et al., 1986). In chickens germline-competent ES cells have been developed and maintained up to 7 d (Pain et al., 1996), but the efficiency of germline transmission (GLT) of cultured cells is very low. The production of chimeras as an intermediate to developing transgenic chickens has been achieved by injecting Stage X blastodermal cells into Stage X recipient blastoderms (Petitte et al., 1990). This paper analyzes the MHC (B* haplotype) antigens in the peripheral blood of chimeric chickens to assess donor line MHC expression in chimeras and to evaluate if the MHC haplotype influences GLT. The genes of the polymorphic EaB blood group locus (Briles et al., 1950) were shown to act as the chicken MHC (Schierman and Nordskog, 1961). The EaB locus is subdivided into a complex of several loci. These include Class I (B-F) and Class II (B-L) genes that are similar to MHC genes of mammalian species and unique Class IV

Abbreviation Key: BR = Barred Plymouth Rock; ES = embryonic stem; FC = flow cytometry; GLT = germ line transmission; HA = hemagglutination; RBC = red blood cells; UG = University of Guelph; WL = White Leghorn.

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ABSTRACT Chimeric chickens were evaluated as an intermediate for development of transgenic chickens. The transfer of Barred Plymouth Rock (BR) blastodermal cells into White Leghorn (WL) embryos results in BR→WL chimeras, and some breeder males generate over 30% germline transmission of the BR genotype to offspring based on a feather-color trait. The objectives of the current study were to 1) identify the MHC (B haplotypes) in resident BR and WL lines, 2) establish that B antigens could be detected and quantified in red blood cells (RBC) of chimeras, 3) establish if there is a correlation in chimeras between percentage of RBC with donor B antigens and percentage germline transmission, and 4) evaluate if the MHC genotype influences chimera development. The RBC agglutination data indicated three B haplotypes were present in each line. The B*2-like, and B*19-like genes were

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MATERIALS AND METHODS Experimental Design Selected B antisera were used to evaluate chimeras from four experiments. First, a retrospective study was made using previously developed BR→WL chimeras that were examined for B haplotypes. Then a prospective BR→WL chimera experiment was conducted to assess if the B haplotype influenced chimera development. Subsequently, we examined the efficiency of incorporation of BR blastodermal cells that were transfected with a lacZ construct into WL blastoderms of different B genotypes. Finally, WL blastodermal cells of specific B genotypes were used

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MJ Research Inc., Waltheim, MA.

to make chimeras in WL recipient embryos of different B genotypes.

Chickens Two lines of chickens obtained from a commercial breeder and maintained as closed breeder flocks at the UG were used. The BR line is homozygous recessive (ii) at the dominant white locus and is used as the donor of BC in the production of chimeras. The WL line is homozygous dominant (II) at the white locus and is used as the recipient embryo (Petitte et al., 1990). A chimera constructed by transfer of BR donor cells to a WL recipient embryo is designated BR→WL. The percentage of black feathers in resulting chimeras has been used as a method to quantify somatic chimerism (Carsience et al., 1993).

Chimera Production Chimeras were developed as described (Petitte et al., 1990, 1993; Carsience et al., 1993). Briefly, donor Stage X blastoderms were obtained from freshly laid fertile eggs, and blastodermal cells were prepared for injection. The development of embryos in freshly laid fertile recipient eggs was compromised by approximately 500 rads of γirradiation 1 to 4 h prior to injection of approximately 200 to 500 blastodermal cells into the subgerminal cavity of the recipient blastoderm. On the third day of incubation, the contents of a viable recipient egg were transferred into the shell of a surrogate egg to enhance production of hatched chicks (Rowlett and Simkiss, 1987). On Day 20 of incubation, the embryos were transferred to trays in a hatching incubator. Hatched putative chimeras and appropriate control chicks were analyzed for the expression of MHC antigens and, where appropriate, for somatic chimerism estimated from the extent of black feather pigmentation.

Evaluation of Germline Status of Putative Chimeras The level of germline chimerism was estimated by mating the adult BR→WL chimeras to BR breeders to determine the percent production of black chicks. In order to identify markers associated with adequate reproductive fitness the germline chimera analyses were limited to chimeras producing 10 or more chicks.

PCR Analyses The DNA samples were prepared by adding 500 µL of autoclaved water to 30 µL of blood and placing the sample in a beaker of boiling water for 8 min. The samples were then cooled on ice and centrifuged for 4 min at 850 × g. Four microliters of the supernatant were added to a 100µL PCR cocktail that included 0.025 U/µL of Taq DNA polymerase, 10mM Tris-HCl pH 9.0, 50 mM KCl, 1.5 mM MgCl2, and 0.2 mM dATP, dTTP, dGTP, and dCTP. Amplification of DNA was accomplished in a PTC100TM programmable thermal controller.4 The presence

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(B-G) genes present in birds (Pink et al., 1977; Guillemot et al., 1988; Miller et al., 1988; Zoorob et al., 1993). The B-G, two B-F (FI and FIV), and two B-L (LI and LII) loci have been linked to the B haplotype. The B haplotype segregates independently of additional Class I (FV and FVI) and II (LIII, LIV, and LV) genes that are in a haplotype termed Rfp-Y (Briles et al., 1993; Miller et al., 1996). In a chicken line segregating for both haplotypes, only the B haplotype acted as the MHC, i.e., determined antigens causing rapid skin-graft rejection and significant mixed lymphocyte responses (Pharr et al., 1996). The B haplotype genes have been associated with disease resistance and vaccinal immunity (Bacon 1987; Lamont et al., 1987; Bacon and Witter, 1994). The identification of B haplotype (as well as other) alloantigens with specific antisera has often been used as a method to quantify donor cells in studies of transplantation tolerance induction with chickens (for example, see Lehtonen et al., 1985). However, to the authors’ knowledge there are no studies that employ B antisera to quantify donor cells as an indicator of chimerism. The current study describes the use of B antisera to identify three B haplotypes in a Barred Plymouth Rock (BR) line and three B haplotypes in a White Leghorn (WL) line. These lines have been utilized, respectively, as donors of and recipients for blastodermal cells in the production of chimeric chickens at the University of Guelph (UG). Selected antisera were then used in flow cytometry (FC) to estimate the percentage of donor BR red blood cells (RBC) in the peripheral blood of chimeras that had been produced prior to the initiation of the present study. Prospective experiments were then conducted to study the establishment of BR or WL donor blastodermal cells of known B genotype(s) in chimeras developed in recipient WL of different B genotypes. An additional experiment tested the efficiency of incorporation of donor BR blastodermal cells in recipient WL blastoderms of different B genotypes by tracking the fate of donor cells transfected with a lacZ reporter gene in early chick embryos.

MHC EVALUATION IN CHIMERIC CHICKENS

Definition of B* Haplotypes by Hemagglutination The B haplotypes in the two UG chicken lines were initially defined by hemagglutination (HA) utilizing alloantisera specific for the B haplotype (Fulton et al., 1996). Conventional B antisera were produced by immunizing WL 15I5 B congenic B-heterozygous chickens with B-homozygous blood. Antisera specific for B-FIV antigens were prepared by immunizing F1 hybrid chickens with transfected syngeneic tumor cells expressing B-FIV molecules (Fulton et al., 2001). The B allelic designations were assigned to UG line chickens based on the specificity of reactivity of the various antisera in HA of RBC. The designation of an allele in a UG line indicates the presence of an antigenic epitope detected serologically in that line and in the B-congenic line having the designated allele. The DNA analyses to define the extent of identity were not performed. Thus, designation of an allele in a UG line, e.g., B21, indicates the antigenic product of an allele is B21-like, but it may not be identical to B21. Antisera were absorbed where needed to remove undesired crossreactivity, e.g., the B13 antiserum was absorbed with B*2/

5 Hans Cheng, Avian Disease and Oncology Laboratory, East Lansing, MI. 6 Bethyl Laboratory, Inc., Montgomery, TX. 7 Beckman Coulter, Inc., Fullerton, CA.

*2 and B*19/*19 RBC from the WL line to remove any negligible reactivity prior to use in FC. Matings of chickens with designated B* alleles were made to produce chicks with specific B* genotypes.

Chicken Matings After blood-typing, breeders were caged according to genotype, and matings were made within WL to produce chicks of known B genotype. To evaluate an influence of B*19 vs. B*21 haplotypes on the production of BR→WL chimeras, WL embryos were obtained from a mating of two males (one B*19/*21 and one B*19/*19) with 22 females (17 = B*19/*21 and 5 = B*19/*19). To evaluate RBC chimerism in WL→WL chimeras, matings were made to produce pedigreed WL fertile eggs homozygous for B*2/*2, B*19/*19, or B*21/*21 or heterozygous for B*2/*21. Ultimately, a few chicks from these matings had B* genotypes differing from pedigree expectations, and so the breeders were blood-typed again after the experiments were established. The B* genotypes of all males and of the B*21/*21 females were substantiated. However, three of the 19 hens first designated B*2/*2 were reclassified as B*2/*21, and so when mated to B*2/*2 males about 5% of the total offspring would be B*2/*21, and when mated to B*21/*21 males about 5% would be B*21/ *21. Also, seven of the 74 hens first designated B*19/*19 were reclassified as B*19/*21, and one was B*2/*19. It is predicted that these hens mated to B*19/*19 males would result in about 95% B*19/*19 chicks as planned, but 5% of all chicks would be B*19/*21 and 0.5% B*2/*19.

FC The RBC were incubated with B alloantibodies having specificity in HA, followed by incubation with fluoresceine labeled goat anti-chicken immunoglobulin.6 The FC procedures described previously (Fulton et al., 1996) were used. The evaluations were done with a Coulter Epics Elite flow cytometer.7 For an unbiased FC evaluation, cells with >2 log channels of fluoresence were regarded as positive for expression on an antigen, e.g., if 5% of an RBC sample from a chicken incubated with B13 antiserum had a log channel fluoresence >2 it was concluded that 5% of the RBC had B13 antigen.

Incorporation of Transfected Blastodermal Cells in Chick Embryos Freshly isolated BR blastodermal cells were transfected by electroporation with plasmid pmiwz (Suemori et al., 1990), which contains a lacZ reporter gene under the control of a Rous sarcoma virus enhancer and tandem promoters derived from Rous sarcoma virus and the chicken β-actin gene, as described (Zajchowski, 1998). Briefly, 2 to 5 × 105 blastodermal cells and 10 µg of plasmid pmiwz were resuspended in 70 µL of cytomix buffer (van den Hoff et al., 1992) in a standard 1-mm gap electroporation

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of DNA in the samples was confirmed by amplification of a 720-bp product from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as described by Clinton (1994). One chicken-line-specific microsatellite was used, i.e., ADL0136, identified hereafter as Microsatellite M. The forward and reverse primers were 5′ TGT CAA GCC CAT CGT ATC AC 3′ and 5′ CCA CCT CCT TCT CCT GTT CA 3′, respectively, and were kindly supplied by Hans Cheng5 (Cheng et al., 1995). Samples were denatured at 94 C for 3 min; followed by 30 cycles at 94 C for 1 min to denature the samples, 1 min at 54 C to anneal the primers; and 72 C for 1 min for elongation of the DNA strand. The 30 cycles were followed by a final elongation 10 min at 72 C. With genomic WL DNA, two bands were present, but when BR DNA was added, three template bands were observed. Therefore an additional band in DNA from a putative chimera indicated the presence of BR chimeric cells. A W chromosome (femalespecific) microsatellite was amplified with the forward primer 5′ CGT GAG AAA AGT GGT AGT TC 3′ and the reverse primer 5′ CCA AAA ATA CCA CCT GTC TC 3′ and resulted in a 276-bp product (Clinton, 1994). Amplification was initiated at 94 C for 3 min; followed by 30 cycles at 94 C for 45 s to denature the samples, 30 s at 54 C to anneal the primers, and 72 C for 1.5 min for elongation of the DNA strand; and a final elongation of 10 min at 72 C. The presence of the 276-bp product in a male putative chimera indicated that he was chimeric for BR female cells.

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Statistical Analyses Parametric traits with continuous values, e.g., the effect of B genotype on percentage black plumage, percentage B13-positive RBC, or percentage black chicks, were analyzed by two-way ANOVA. Non-parametric categorical traits, i.e., the effect of B* genotype on percentage of chicks with DNA positive for the M or W probes, were analyzed by Pearson chi-squared tests. A significance level of P < 0.05 was required for all tests. These analyses, as well as Spearman-rank correlation coefficients (RS), were computed using the JMP statistical package (SAS Institute, 1995). For the experiment examining the incorporation of transfected blastodermal cells in recipient WL blastoderms of various B genotypes, the frequency of intraembryonic chimerism was analyzed using a categorical model. The level or extent of intra-embryonic chimerism was analyzed by ANOVA following a log transformation of the data.

RESULTS AND DISCUSSION Evaluation of B Genotypes in UG Chickens WL. One or more antisera specific for the B2, B5, B12, B13, B15, B19, or B21 antigens, which have variable specificity or cross-reactivity for other antigens depending on

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BTX Division, Gentronix, Inc., San Diego, CA.

the B genotype of the antibody producing chicken, were used for HA of RBC from 20 adult males. Only three B alleles were detectable in the WL males (B*2, B*19, and B*21) resulting in three homozygous (B*2/*2, B*19/*19, and B*21/*21) and three heterozygous (B*2/*19, B*2/*21, and B*19/*21) genotypes. The WL females were then tested by HA using the most informative antisera that had very little or no cross-reactivity, i.e. B2 #1397D (comparable to #1400F, Fulton et al., 1996), B19 #1396H (comparable to #1401O, Fulton et al., 1996) and BFIV21 #E2601 (Fulton et al., 2001). The gene frequencies for both sexes in 255 WL were: B*2 = 0.33, B*19 = 0.58, and B*21 = 0.09. The observed B genotype frequencies were close to expectation based on calculations from the observed gene frequencies, and it was concluded that three B alleles were definable in the UG WL line using standard B alloantisera. Within the WL line the B2, B19, and B21 antisera had comparable specificity for RBC in HA and FC. BR. B antisera were also used in HA to assess RBC from 20 BR males. Three B alleles were identified in the BR males (B*13, B*15, and B*21), resulting in six genotypes (B*13/*13, B*15/*15, B*21/*21, B*13/*15, B*13/*21, and B*15/ *21). Subsequently, informative antisera that had very little or no cross-reactivity were used in HA of RBC from BR females, i.e., BFIV13 #E2595, B15 #492K and BFIV21 #E2601 (Fulton et al., 1996, 2001). The gene frequencies in 251 BR males and females were B*13 = 0.14, B*15 = 0.50, and B*21 = 0.36. The observed B genotype frequencies were close to expectation based on the calculations from the observed gene frequencies, and it was concluded that three B alleles were definable by HA in the BR line using standard B antisera. However, in the BR line, the B13 and B15 antisera had different specificity in FC than in the HA assays. In FC the B13 and B15, antisera crossreacted to detect only one B antigenic type. The very sensitive FC assay apparently detected an epitope common to B13 and B15 antigens that was undetectable by HA. The B13 antiserum was specific for the B-FIV molecule, and so a common epitope must exist in the BR B13like and B15-like B-FIV molecules. Therefore, the level of BR chimerism in BR→WL chimeras was analyzed in FC using only the B13 antiserum.

FC Analysis of RBC Mixtures and Chimeras RBC Mixtures. To establish the ability of an antiserum to detect RBC of a specific B antigenic type in FC, several proportions of two types of RBC from adult chickens were mixed. In Figure 1, mixtures of various proportions of BR B*13/*13 RBC were made with WL B*2/*2 RBC (0, 10, 50, 90, and 100% B*13/*13 RBC). After FC analysis, the percentage of cells with a log channel fluoresence of >2 channels detected by the B13 antiserum was close to expectation (0, 14, 49, 93, and 100%). Examples of FC Analysis of Chimeras. The BR→WL chimeras were produced using a pool of blastodermal cells from approximately 16 to 24 BR fertile eggs of undefined B genotype. Based on the BR B allele frequencies,

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cuvette and subjected to three 99 µs2 wave electrical pulses with a field strength of 2.0 kV/cm, delivered at 1s intervals by a BTX ElectroSquarePorator T820.8 Electroporation was performed at room temperature. A transfection efficiency of approximately 70% and cell viability of approximately 50 to 60% were consistently achieved under these conditions. Approximately 500 transfected blastodermal cells were injected into irradiated WL blastoderms of defined B genotypes as described above, and eggs were incubated for 72 h. At this time, viable chick embryos were recovered in order to measure incorporation of transfected donor cells by histochemical staining for the presence of exogenous β-galactosidase activity, as described by Zajchowski (1998). Briefly, embryos were dissected from the yolk, washed in PBS to remove excess yolk, and fixed in cold 4% paraformaldehyde for 20 min. The fixative was removed and embryos were washed three times in PBS and then stained for 1 h at 37 C in 0.5 mg/mL X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 2 mM MgCl2 in PBS, pH 7.4. The presence of blue (β-galactosidase-positive) cells within intra-embryonic tissues was then scored by counting the number of blue cell foci (where one cell focus contained 1 to 5 cells). Control embryos that had not been injected with transfected cells showed no blue staining when similarly treated.

MHC EVALUATION IN CHIMERIC CHICKENS

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FIGURE 1. In flow cytometry, a B13 antiserum did not react to White Leghorn B*2/*2 red blood cells (RBC) (A), but the percentage of detectable RBC was proportional to the percentage B*13/*13 RBC added in a mixture, e.g., 10% (B), 50% (C), 90% (D), or 100% (E). Vertical = number of RBC; horizontal = log mean channel of fluorescence from the flow cytometer.

it was estimated that 87% of the approximately 200 blastodermal cells inoculated into each WL recipient would contain B13 or B15 detectable with B13 antiserum. Figure 2 illustrates the detection of 0% (Figure 2a), 13% (Figure 2b), 68% (Figure 2c), and 81% (Figure 2d) BR RBC in young adult BR→WL chimeras using B13 antisera. Thus, the B13 antiserum could detect BR chimerism in BR→WL chimeras. In the WL protocol, blastodermal cells were exchanged between WL chickens differing for B genotypes, and the donor RBC were identified by FC. Figures 3 and 4 illustrate the detection of 0 or 44% B*19/*19 RBC in a B*19/ *19→B*2/*21 chimera, or B*19/*19→B*21/*21 WL chimera.

FIGURE 3. Chicken No. 6550 was a 2-wk-old B*19/*19→B*2/*21 White Leghorn chimera. (A) 0% of red blood cells (RBC) react with B19 antiserum, indicating No. 6550 contained no donor RBC; (B) an antibody detecting B2 reacted to 96% of RBC at this young age. Vertical = number of RBC; horizontal = log mean channel of fluorescence from the flow cytometer.

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FIGURE 2. Examples of different levels of chimerism in Barred Plymouth Rock→White Leghorn chimeras based on reactivity of B13 antiserum to red blood cells (RBC) from adult chimeras. The percentage of RBC reacting to B13 ranged from A (No. 4975) = 0%, B (No. 4959) = 13%, C (No. 4169) = 68%, and D (No. 4197) = 81%. Vertical = number of RBC; horizontal = log mean channel of fluorescence from the flow cytometer.

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In the converse chimera experiment, 11% of the RBC were reactive to a B21 antiserum in a B*2/*21→B*19/*19 WL chimera (Figure 5). Thus, FC also detected chimeric donor RBC in 2-wk-old WL chimeras.

Influence of MHC and Other Traits on Germline Transmission in Existing BR→WL Chimeras The existing adult BR→WL male (47) and female (80) chimeras were analyzed by FC with B13 antiserum to establish the percentage of donor RBC in their blood. Many chimeras RBC were repeatedly analyzed, and the results were highly reproducible. In the males there was

FIGURE 5. Chicken No. 6582 is a 2 wk old B*2/*21→B*19/*19 White Leghorn chimera. (A) 11% of red blood cells (RBC) are reactive to B21 antibody indicating No. 6582 is 11% chimeric for the donor RBC; (B) 48% of RBC are reactive with B19 antibody at this young age. Vertical = number of RBC; horizontal = log mean channel of fluorescence from the flow cytometer.

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FIGURE 4. Chicken No. 6488 was a 2-wk-old, B*19/*19→B*21/*21 White Leghorn chimera. (A) 44% of red blood cells (RBC) reacted with B19 antiserum, indicating No. 6488 was 44% chimeric for the donor RBC; (B) B21 antibody reacted with 40% of the chimeras RBC at this young age. Vertical = number of RBC; horizontal = log mean channel of fluorescence from the flow cytometer.

a non-significant negative correlation between percentage B13 RBC in blood and number of chicks they produced (RS = −0.21, P > 0.13). However, in females the negative correlation between the percentage B13 RBC in blood and the number of chicks produced was highly significant (RS = −0.38, P < 0.0009). Indeed, six of 11 (54%) hens that failed to produce 10 chicks had ≥3% B13 RBC in their blood (data not shown). The data for reproductive, existing chimeric breeders that produced ≥10 offspring was evaluated for the traits under analysis. The 44 chimeric males had a higher percentage of germline transmission than the 69 females, and therefore the data for various traits were tabulated and analyzed within sex (males are in the upper half of Table 1 and females are in the lower half). The existing adult BR→WL chimeras were each ascribed one of six possible WL B genotypes based on HA tests (Table 1, Column 2). Only four males and one female were B*21/*21 (Table 1, Column 3). In the male chimeras, there was an association between the B* genotype and percentage of black feathers in their plumage, i.e., the B*21/*21 male breeders were significantly blacker than males in other B* genotype groups except for B*2/*2 males (Table 1, Column 4). In the females a similar trend was observed, but since the percentage in B*21/*21 was based on one hen, it was not involved in the statistical analysis. Blood containing ≥3% B13 RBC was observed with only 39% of the males and 20% of the hens. However, 27% of all males and 17% of all females had >50% B13-positive RBC (data not shown). Given this variability in B13 RBC detection, the B* genotype was associated with the percentage of RBC reactive to B13 antiserum in FC in both sexes (Table 1, Column 5). The B*21/*21 males had a significantly higher percentage of RBC reactive with B13 antiserum than males lacking B*21, and the B*21 heterozygotes had intermediate percentages of reactivity. A similar trend was also observed with the females. In the B*21/*21 male chimeras, 75 and 100% were positive for the BR DNA using the M and W microsatellite probes, respectively (Columns 6 and 7), and 60% of the B*2/*21 males contained BR DNA detectable with both probes. In contrast, the microsatellite probes detected BR DNA at a significantly lower frequency in male chimeras of the other B* genotypes (B*2/ *2, B*2/*19, B*19/*19, or B*19/*21). Most interestingly, there was a significant association between the B genotype and germline transmission in males (Table 1, Columns 8 and 9); 35% of 180 offspring from B*21/*21 males had black plumage compared to ≤6% of similar numbers of offspring from the other B genotype groups. In male chimeras, the production of black chicks was significantly correlated with the percentage of B*13 RBC in blood (RS = 0.44, P = 0.003) and percentage of black feathers in the plumage (RS = 0.38, P = 0.01), but the correlation with the M microsatellite was not significant (RS = 0.30, P = 0.10). However, in female chimeras the first two of these correlations were low and nonsignificant (RS = 0.04, P = 0.74; RS = 0.12, P = 0.22, respectively). Given the significant correlation between percentage B13 RBC and germline transmission by male BR→WL

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MHC EVALUATION IN CHIMERIC CHICKENS TABLE 1. Association of the MHC with somatic or germline transmission traits in existing BR→WL chickens Chimera germline transmission4

Somatic trait in chimera B* genotype group Sex

Producing ≥ 10 chicks (n)

Black plumage1 (%)

B13 + RBC2 (%)

BR DNA M probe3 + (%)

BR DNA W probe3 (%)

Number chicks (X)

Black chicks (%)

2/2 2/19 2/21 19/19 19/21 21/21 2/2 2/19 2/21 19/19 19/21 21/21

5 13 5 10 7 4 10 23 4 26 5 1

51.8ab 32.6b 36.4b 35.0b 39.2b 82.0a 25.0a 32.6a 43.a 29.1a 40.0a 90.0

2.8c 1.0c 49.2ab 24.7bc 27.8bc 87.8a 16.8ab 4.1b 40.5a 5.1b 21.2ab 75.0

20abc 15bc 60ab 0c 14bc 75a Not done ... ... ... ... ...

20bc 15bc 60ab 0c 14bc 100a Not done ... ... ... ... ...

203 182 164 172 242 180 71 92 74 87 77 63

3.0b 5.2b 6.0b 5.1b 3.1b 35.0a 0.2a 1.2a 0a 1.5a 4.0a 0

a-c Within sex, the values of a trait in a column with no common superscript are significantly different (P < 0.05; not calculated for the single B*21/*21 female). 1 The percentage of black feathers in plumage of each chimera was recorded at hatch, and the group mean is given. 2 The percentage of red blood cells (RBC) reactive to B13 in flow cytometry was defined in each adult chimera, and the group mean is given. 3 DNA was obtained from each chimera and evaluated with the M or W microsatellite probe to detect Barred Rock (BR) DNA. The percentage of the group positive for BR DNA is given. 4 Each chimera was mated to a White Leghorn breeder. Group means are given for the number of chicks produced, and the percentage of black chicks, i.e., germline transmission.

chimeras in Table 1 the average percentage germline transmission by the 17 males with ≥3% B13 vs. all 44 males was computed. Those with ≥3% B13 RBC had an average of 14.6% germline transmission, which was almost twofold higher than an average of 7.4% germline transmission by all 44 males.

Influence of MHC and Other Traits on Germline Transmission in BR→WL Chimeras Produced by B*19/*21 WL Parents The BR→WL chimeras were produced using pooled BR blastodermal cells injected into blastoderms of fertile eggs from WL parents segregating for B*19 and B*21. At 2 to 4 mo of age the chimeras RBC were analyzed by FC using B13 and B21 antisera to establish the percentage of donor RBC in their blood. In the B*19/*19 chickens, the RBC of chickens reactive with B13 antiserum were also reactive with B21 antiserum, and the percentage of RBC reactive with either antibody was consistently similar (data not shown). Approximately half of all chimeras that produced 10 or more chicks (51% of 43 males and 44% of 36 females) had no RBC reactive to B13 antiserum. Another 19% of the males and females had 1 to 3% of RBC reactive with B13, leaving only 30% of males and 37% of females with clear evidence for donor RBC in the blood. Still, 16% of male and 6% of female chimeras had predominantly (50 to 70%) B13-reactive RBC. The chimeric males had a higher percentage of germline transmission than females, and therefore data for analyzed traits were tabulated within sex (males are in the upper half of Table 2 and females are in the lower half). The putative chimeras were evaluated for percentage black feathers in plumage at hatch and were blood-typed

at 3 wk of age. Similar proportions of B*19/*19 and B*19/ *21 chickens were identified, but B*21/*21 chickens were less frequent due to the B genotypes of the parents. The B genotype of the chimeras was not associated with the percentage of black feathers in the plumage (Table 2, Column 4). Although there was a trend for the percentage of RBC reactive to B13 to be greater in the B*21/*21 than B*19/*19 males and females, the differences were not significant (Column 5). The detection of BR DNA utilizing the M microsatellite probe was similar in males of different B genotypes. However, in females, BR DNA was detected more frequently in the B*21/*21 than B*19/*21 genotype (P < 0.05), but the low frequency in B*19/*19 females was not significant (Column 6). The percentage of germline transmission for males was remarkably good (Columns 7 and 8). There was a trend for germline transmission to be higher in males and females of the B*21/*21 and B*19/*21 genotypes compared to the B*19/*19 genotype, but the differences were not statistically significant. In male chimeras the production of black chicks was significantly correlated with the percentage of B*13 RBC in blood (RS = 0.56, P < 0.001), as well as the percentage of black feathers in the plumage (RS = 0.54, P < 0.002) and presence of the M satellite (RS = 0.42, P < 0.001). However, in female chimeras these correlations were lower with limited or no significance (RS = 0.12, P = 0.47; RS = 0.33, P = 0.05; and RS = 0.31, P = 0.07, respectively). Given the moderate correlation between percentage B13 RBC and germline transmission by male BR→WL chimeras, the average percentage germline transmission by the 13 males with >3% B13 RBC vs. all 43 males was computed. Males with >3% B13 RBC had an average of 48.3% germline transmission, which was 162% higher than an average of 29.8% germline transmission by all males.

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Male Male Male Male Male Male Female Female Female Female Female Female

B* genotype

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TABLE 2. Association of the MHC with somatic or germline transmission traits in BR→WL chickens produced by White Leghorn parents segregating for B*19 and B*21 Chimera germline transmission4

Somatic trait in chimera B* genotype group Sex Male Male Male Female Female Female

B* genotype

Producing ≥ 10 chicks (n)

Black plumage1 (%)

B13 + RBC2 (%)

BR DNA M probe3 + (%)

Number chicks (X)

Black chicks (%)

19/19 19/21 21/21 19/19 19/21 21/21

16 17 10 14 18 4

46.9a 49.6a 31.0a 52.9a 50.6a 50.0a

7.2a 12.5a 11.8a 7.9a 7.8a 17.2a

37.5a 23.5a 20.0a 28.6ab 22.2b 75a

146 150 188 57 56 40

19.8a 36.0a 35.2a 1.9a 13.8a 3.8a

Within sex the values of a trait in a column with no common superscript are significantly different (P < 0.05). The percentage of black feathers in plumage of each chimera was recorded at hatch, and the group mean is given. 2 The percentage of red blood cells (RBC) reactive to B13 in flow cytometry was defined in each young adult chimera and the group mean is given. 3 DNA was obtained from each chimera and evaluated with the M microsatellite probe to detect Barred Rock (BR) DNA. The percentage of the group positive for BR DNA is given. 4 Each chimera was mated to a White Leghorn breeder(s). Group means are given for the number of chicks produced, and the percentage of black chicks, i.e., germline transmission. a,b 1

Pedigreed fertile eggs were obtained from WL matings established to produce six possible B genotypes. As described in the chicken matings, some hens were not B homozygous as initially defined (see above), which led to a potential of 5% unplanned B heterozygosity in the pedigree eggs of all except the B*21/*21 group. The irradiated WL embryos were injected with BR blastodermal cells transfected with a lacZ reporter gene. After 72 h, the embryos were stained with X-gal to detect transfected donor cells expressing β-galactosidase. The frequency of embryos containing at least one transfected cell focus ranged from a low of 36% for the B*21/*21 group to a high of 51% for the B*2/*2 group, but no statistically significant differences were observed (P = 0.6858; Table 3). Similarly, the number of transfected cell foci present per embryo was not significantly different among recipient blastoderms of different B genotypes (P = 0.9538; Table 3). Up to 43 transfected cell foci were observed in the intraembryonic tissues of a single embryo, but the average number of foci ranged from about three to four in chimeric embryos of all B genotypes. It was concluded that the WL B genotype did not influence the establishment of

BR blastodermal cells in BR→WL chimeras at 72 h postinjection.

Use of B-Haplotype to Evaluate Chimerism in WL The WL chimeras were produced by exchanges of blastodermal cells between pedigreed fertile eggs that were designed to be B*19/*19→B*2/*21 or B*21/*21, or B*2/ *21→B*19/*19 or B*2/*19. At 2 wk of age the potential chimeras were analyzed for B genotype by HA, and an estimate of chimerism was defined by FC using B2, B19, or B21 antiserum. The percentage of chickens of different B genotypes identified by HA was within expectation based on the parental genotypes. Figure 6 provides a summary of the FC results for RBC from 130 control and putative chimeras. Note that each B antiserum reacted to less than 5% of RBC in the negative controls for that antiserum. For example, the B19 antiserum reacted with less than 2% of B*2/*21 or B*21/*21 RBC (lower series, fourth and seventh bars up, respectively). By contrast, in positive controls the B2 antiserum reacted to 93% of B*2/*21 RBC (top series fourth bar up). Also, the B21 antiserum reacted to 42% of B*2/*21 RBC and 50% of the B*21/*21 RBC (middle series, fourth and seventh bars up, respectively), and the B19 antiserum to 52% of B*19/*19 RBC (lower series, bot-

TABLE 3. The effect of B genotype of recipient White Leghorn blastoderms on the efficiency of incorporation of transfected Barred Rock (BR) donor blastodermal cells (BC) in 72 h chick embryos B genotype of recipient blastoderm B*21/*21 B*19/*19 B*2/*2 B*19/*21 B*2/*21 B*2/*19

Total number of embryos

Number of embryos containing transfected BR BC (%)

Average number of transfected cell foci per embryo

22 65 35 42 36 44

8 (36%) 26 (40%) 18 (51%) 19 (45%) 15 (42%) 21 (48%)

3.6 3.4 2.9 2.9 3.9 3.5

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Efficiency of Incorporation of BR Blastodermal Cells into WL Blastoderms of Different B Genotypes

MHC EVALUATION IN CHIMERIC CHICKENS

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reacted to 50% of 44 B*2/*21→B*19/*19 and 100% of 2 B*2/ *21→B*2/*19 putative chimeras.

Utility of Blood-Typing for MHC Expression in Evaluation of Chicken Chimeras

tom bar). Thus, the B2 antiserum had more reactivity than the B19 or B21 antiserum, probably due to a lower expression of the requisite B19 and B21 antigenic epitope(s) at 2 wk of age or a higher avidity of the B2 antiserum. The mean percentage of B*19/*19 RBC in B*19/ *19→B*2/*21 or B*21/*21 recipient chimeras was 18 to 20% (lower series second and third bars up, respectively). This result was 6% higher than the mean percentage of B*2/ *21 RBC in B*2/*21→B*19/*19 recipient chimeras (12%, middle series, fifth bar up using B21 antiserum; and 13%, top series, fifth bar up using B2 antiserum). From a broader perspective, over half of the putative chimeras in this WL model had ≥3% donor-type RBC (data not shown). Thus, the B19 antiserum reacted to 64% of 28 B*19/*19→B*2/*21 and 100% of five B*19/*19→B*21/*21 putative chimeras. Alternatively, B2 or B21 antiserum

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FIGURE 6. The red blood cells (RBC) of each 10- to 13-d-old White Leghorn control and chimeric chick were mixed with B2, B19, or B21 antiserum, and the percentage of RBC with a log channel fluorescence (LCF) value of >2 was defined by flow cytometry. Bars of different patterns record the mean percentage of RBC with an LCF >2 for each control and chimeric group. The upper series of bars summarize mean reactivity of each group to B2 antiserum, the middle series reactivity to B21 antiserum, and the lower series reactivity to B19 antiserum. a-c Means with no common letter within a series are different (P < 0.05). The number of chickens in each group is given at the end of each bar in the lower (B19) series.

Defined antisera were shown to recognize three B haplotypes in HA of RBC from chickens of the UG BR line. Three B haplotypes were also identified in chickens of the UG WL line. A B*21-like haplotype was present in both lines, whereas B*13-like and B*15-like haplotypes were unique for the BR line, and B*2-like and B*19-like haplotypes were unique to the WL line. The B genotype frequencies observed in each line were within expectation based on the gene frequencies of each B haplotype. Moreover, when accurately identified breeders were mated, they only produced chicks of the expected B genotypes. Similar B haplotypes are common in WL chickens (Bacon, 1987), but their presence in BR chickens is unknown. It was concluded that B antisera detects differences between UG chicken lines used as donors and recipients in the production of chimeric chickens and that they are potentially useful in identifying donor cells in chimeras. A marker with expression on the surface of all nucleated cells is desirable for evaluating chicken chimeras. The molecules produced by the B-FIV gene have this characteristic and are expressed on RBC. The B13 and B21 antisera were specific to B-FIV molecules (Fulton et al., 2001). The B2, B15, and B19 antisera must also contain B-FIV antibodies because they react to white blood cells that normally do not express BG antigens, as well as to RBC that express B-FIV and BG antigens (Fulton et al., 1996). The B antisera were shown to react with specificity in FC, and when mixtures of the BR B*13/*13 and WL B*2/ *2 RBC were analyzed they could detect with precision the percentage of each type RBC present. Therefore selected B antisera were used in FC to evaluate several experimental groups of chimeras. The correlation of several traits with germline transmission was of interest in the BR→WL chimeric males. Significant correlations existed between presence of donor B13 RBC and germline transmission (RS = 0.44 and RS = 0.56 in Experiments 1 and 2). If this moderate correlation is confirmed in lines of interest, then RBC of putative chimeras could be screened in FC with specific antiserum for the donor line B type by 4 wk of age. About twothirds of the BR→WL chimeras in Experiments 1 and 2 lacked evidence for donor RBC, and therefore one could select and rear to adulthood for test mating only those chimeras having the best potential for germline transmission. Slightly lower significant correlations were seen between black feather plumage and germline transmission (R = 0.38 and 0.54 in Experiments 1 and 2, respectively). This quantitative somatic trait was the only one previously studied in the chimeras but was only a fair indicator of germline transmission (Carsience et al., 1993). A non-quantitative trait was also defined in the chimeras, i.e., analysis of DNA for the presence of BR DNA using microsatellite probes M and W. The chimeric

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(Table 3). If B* genotype influenced incorporation of BR cells, it is unlikely that incorrect identification of 5% of the WL blastoderms would have significantly influenced the results. Moreover, although the transfer of pooled WL B*19 blastodermal cells might have contained 5% B*2 or B*21 blastodermal cells, this finding would seem irrelevant as the cells were injected into B*2/*21 or B*21/*21 blastoderms. Likewise, the blastoderms from B*2/*21 matings might have contained 5% more B*21 cells than expected, but this is unlikely to have affected results when injected into B*19/*19 blastoderms.

Influence of MHC on Germline Transmission A major goal of the BR→WL chimera research at the UG has been to develop methods to produce chimeras with a high proficiency for germline transmission. It is therefore of interest to evaluate if the chicken MHC might influence development of chimeras with optimal germline transmission. Class I MHC antigens are expressed in the earliest stages of embryogenesis in mice based on a very sensitive PCR technology (McElhinny and Warner, 1997) and in chick embryos at 6 d using Northern blots (Dunon et al., 1990). Most interestingly, evidence indicates there are differences in the level of MHC Class I gene expression in chickens of different haplotypes (Briles, 1964; Kaufman and Venugopal, 1998), although this is not established using BF-IV antiserum. In the data from previously developed BR→WL chimeras, there was significant evidence that males assigned the B*21/*21 genotype had a higher level of germline transmission than those of other B genotypes involving B*2 and B*19 in the WL line. Indeed, 35% of the chicks produced by chimeric males assigned the B*21/*21 genotype were donor derived, whereas only 3 to 6% of the chicks produced by males of other B genotypes were donor derived. To establish a B haplotype influence on development of germline chimeras, two additional experiments were done using fertile eggs from pedigree mated WL chickens. One experiment involved transfection of a reporter lacZ gene into BR blastodermal cells that were injected into blastoderms of six different WL B genotypes. After 72 h there were no statistical differences in the frequency or extent of incorporation of β-galactosidase expressing cells in blastoderms from the six different Bgenotype groups, indicating that B haplotype did not influence initial acceptance or growth of donor blastodermal cells in recipient embryos. However, this result does not rule out the possibility that the B haplotype could affect chimera formation at later stages of development. The second experiment involved production of new BR→WL chimeras using fertile eggs that were segregating for B*19 and B*21. Although there was an anticipated trend for the B haplotype to influence germline transmission, the differences were not statistically significant, i.e., in males B*21/*21 = 35.2%, B*19/*21 = 36%, and B*19/ *19 = 19.8% germline transmission. Thus, neither of the current extensive experiments provided significant data

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groups with the highest average percentages of B*13 RBC were also the ones having the highest percentages positive by M and W microsatellite analysis. However, the correlation between M microsatellite reactivity and germline transmission was relatively low (RS = 0.30 and 0.42 in Experiments 1 and 2, respectively). In the chimeric hens the correlations for these traits and germline transmission were generally low or non-significant. The low correlations in hens might have been attributable to the inability of many with higher percentages of B13 RBC and black feathers in their plumage to produce fertile eggs resulting in 10 or more chimeric chicks. This evidence for a detrimental affect of a high level of chimerism on chick production was not anticipated based on earlier analyses (Kagami et al., 1995). Some investigators are beginning to evaluate WL→WL chimeras, but methods for the detection or quantitative analysis of chimerism are not published. Matings of WL of defined B haplotypes were developed in order to produce chicks that were B*2/*21 or B*21/*21 vs. B*19/*19. Following transfer of blastodermal cells between embryos, the putative chimeras were hatched for use in a study to be reported separately and at 10 to 14 d of age RBC were analyzed by blood-typing and FC. At 2 wk, B antigens approach adult levels of expression, although the intensity, i.e., mean log channels fluoresence, may differ between B haplotypes (Henry Hunt, 2000, Avian Disease and Oncology Laboratory, East Lansing, MI, personal communication). In B*19/*19→B*2/*21 chimeras, the B19 antiserum detected ≥3% donor RBC in about two-thirds of chimeras, with an average of 18% in each chimera. The level of chimerism was lower in B*2/*21→B*19/*19 chimeras using the B21 antiserum, as only one-half of the chimeras had donor RBC, and the average level of donor RBC was 13%. Although the difference in percentage chimerism appeared subtle, it indicated chimerism was 33% higher in B*19/*19→B*2/*21 chimeras than B*2/*21→B*19/ *19 chimeras. It was concluded that B antisera can be used in FC of RBC to detect and quantitate a relatively high level of chimerism in WL chimeras at 2 wk of age. Moreover donor-type RBC were detectable in over onehalf of the WL→WL putative chimeras compared to only about one-third of the BR→WL putative chimeras. This finding suggests that production of chimeras may be more effective if conducted within one strain, provided one has a trait to identify the chimerism. In the chicken matings section it was noted that inaccurate blood-typing of several hens resulted in unexpected B* genotypes in a few progeny. These inaccurate matings were not used for data in Tables 1 or 2 but were in effect for data in Table 3 and Figure 6. Five percent of WL progeny from B*2/*2 matings could have been B*2/*21, and 5% of progeny from B*2/*21 matings could have been B*21/*21. Moreover, 5% of B*19/*19 progeny could have been B*19/*21, and 0.5% could have been B*2/*19. However, these inaccuracies are unlikely to have influenced analysis of the results. Ninty-five to 100% of the WL blastoderms of all genotypes injected with BR lacZ transfected blastodermal cells should have been correct as tabulated

MHC EVALUATION IN CHIMERIC CHICKENS

General Conclusions Three conclusions are evident from the present study of MHC antigens in chimeric chickens produced by exchanging blastodermal cells between freshly laid eggs. These are 1) the B haplotype is an excellent marker for identifying and quantifying donor RBC in the peripheral blood of chicken chimeras, even within a strain, 2) the presence of donor RBC antigens may be used to select chimeras for growth to adulthood to enhance efficiency of germline transmission in breeders, and 3) to accurately assess the role of the B genotype on germline transmission, it is essential to use donors that do not have B haplotypes in common with those of the recipient. Unfortunately, in the BR→WL chimera model analyzed here, B*21-like genes were present in the BR and WL lines. Therefore it was impossible to conclude if B*21 leads to greater development of chimeras. However, data from the WL experiment also suggested that the B*21 haplotype in a recipient may result in a higher production of chimeras. Further research is needed to establish if the MHC influences development of chimeric chickens capable of germline transmission.

ACKNOWLEDGMENTS The authors are grateful to Kim Brand (University of Guelph) for extensive and excellent assistance with bloodtyping, artificial insemination, and careful hatching of chimeric chickens. The flow cytometry tests were conducted with advice from Evelyn Young and Henry Hunt (ADOL) and the excellent assistance of Jan Brazolot (University of Guelph). The authors thank Nissim Yonash (ADOL) for statistical advice and Hans Cheng (ADOL) and Ann Gibbins (University of Guelph) for helpful editorial comments. This work was supported in part by the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture, Food and Rural Affairs.

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