Chimeric drift in allophenic mice

CELLULAR

33, 412-422

IMMUNOLOGY

Chimeric Analysis

of Changes

(1977)

Drift

in Allophenic

in Red Blood Cell

and White

in C57BL/6eA THOMAS

Department

J.

STEPHENS,

of Biochemistry

JUDITH

Mice Blood Cell

Populations

Mice

L. MCIVOR, AND CAROL M. WARNER ’

atid Biophysics,

Iowa State University,

Ames, Iowa 50011

Received May 27,1977 Fifteen allophenic mice of the type C57BL/6 ++ A were quantitatively analyzed for changes in their peripheral white blood cell composition and hemoglobin composition with age. It was found that 7/15 or 47% of the mice showed significant changes, in one or the other of these parameters. The seven mice termed “chimeric drift,” showing chimeric drift were classified as unstable chimeras, as opposed to the eight apparently stable chimeras. Chimeric drift was observed in the direction of either parental type, or back and forth, and was found to be independent of the coat color, age, or sex of the mouse. There was an excellent correlation of peripheral white blood cell and hemoglobin compositions of the stable chimeras. However, the unstable chimeras often showed a marked discordance of these two markers.

INTRODUCTION Allophenic mice may be produced by the fusion of two eight-cell embryos (l-3) or by the injection of a single cell of one mouse strain into the inner cell mass of a blastocyst of a second strain (4). These mice provide an interesting immunological test system because of the apparent tolerance of the two different parental cell types. The mechanism of tolerance in the adult animals is unknown. Experiments attempting to elucidate the mechanism of tolerance, including those supporting the forbidden clone theory and those supporting peripheral mechanisms such as serumblocking factors or suppressor cells, have recently been reviewed by McLaren (5). Several laboratories have reported conflicting results, so it is not clear if more than one mechanism exists or if the use of different strain combinations and experimental protocols has led to the different experimental results. Regardless of the mechanism of tolerance, one important question which remains to be resolved is the stability of the chimeric state during the lifespan of the allophenic mice. Mintz and Palm (6, 7) first suggested that allophenic mice may not be completely stable chimeras, but may show changes in their cellular composition with age. Additional support for this concept comes from the experiments of Wegmann and Gilman (S), Barnes et al. (g-11)) and West (12). All of these previous

studies

have

been of a qualitative

nature.

The present study was undertaken to quantitate changes in the hemoglobin and 1 To whom requests for reprints

should be addressed. 412

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0008-8749

CFIIJIERIC

DRIF’I

Th-

AI.I~OI’Il

I’NI(‘

11 l(‘l<

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peripheral white blood cell compositions of allophenic mice as a function of the age of the mice. The strains C57BL/6 and ,‘i Ivere chosen for the production of tile allophenic animals because of the known genetic differences in their H-2 antigens = single ; (C57BI,/6 = N-Z”; A = H-2”) and in their hemoglobin types (C57BL,/6 A = diffuse) (13). We report here the quantitative analysis of the degree of chimerism in the peripheral white l~loocl cell composition and hemoglobin conq)o~itim of 15 C57B1,/6 ts A allophenic mice at three tlifierent times in their li\-vs.

hlicr. C57BL/6, A, and SJL mice were purcl~asetl from the Jackson Lalmratories, Bar Harbor, Maine. ( ,4xSJL)F1 hybrids were bred in our laboratory. CF1 mice wre purchased from Charles River, Portage, Illich. Allophenic mice were produced as described previously (14, 15 ) llriefly, eightcell embryos were collected iron1 superovulatetl C5713I,/O anti A female nlicc, the anti the eiiibryc~s Lvere nmhaiiicallq zona pellucida was removed with l’ronase, lwmq~etl together at 37°C ant1 were allowed to culture for 23-36 hr in all atnlosphere of 5 “/(; CO, in air. The blastocysts \vere then transferred to l)se~“lol)regnant CFl mice and were allowed to come to term. against C‘.57E11./6 Prrpamtio~~ of atztis~ru1r2 crrftl ryfoto.ririty frstirfg. Antisernim spleen cells was prepared by the method of Ratchelor (lh), using either .\ 01 \vad (A4xSJL)F1 1i yl mc 1 mice as the recipients of the spleen cells. This antiserum used to test peripheral white blootl cells u&h \vere isolatetl l)y the I;icoll-H~l);tcliIc ( 17) gradient centrifugation method. The specificity of the autiserunl ~~1s testy<1 against known mixtures of C57BL/6 and A peripheral bvhite blood cells. ‘1‘1~ :tntiwhite blood cell couqmsitic111 serum was then used to test the unknomm peripheral of the allophenic mice. The isolated peripheral white l~lootl cells I\-ere susl~endetl in either I,-15 or RPM1 1640 medium (Gibco) containing 10% heat-illactivatrtl fetal calf serum, at a concentration of 1-6 x 10” cells/ml. In a typical assa!., 10 P1 oi the cell suspension was mixed with 10 ~1 of the approl~riate antiserwm dilution. Tlirn, IO ~1 of a 1 : 2 dilution of guinea pig sertm (lliles) was adtled as the source of COI,,1,leiiient. The mixture was allmvetl to incubate at 37°C for 1 hr after \\-liicli tillle 10 ~1 of a O.-F% trypan blue solution was added (1X). ;jfter 3-S min, ;t niinimtinl 0i ZOO cells \vas assessed iiiicroscol~ically for \-ial)ility. livery assa>~ \vas l)erfornletl at least in tluplicate. Prrparatiorr of Izrrrlo(/lobirl. Hemoglobins from the C571<1,/6. ;I, alit1 :lllol)llellic nlice \vere collected using the modified method of Clegg and Schroetlvr ( 10 ). I’,ricfly, 0.10 nil of whole blood was collected froll~ the orbital venous siillls 0f c;tch niome ant1 \vas rapidly mixed with 0.05 ml of 3.2% sotliunl citrate (l)[ 1 5.5 j ;~t 4°C. Murine erythrocytes were then washed in O.%c/, saline (pH 7.0) ;111(1\vcrc centrifuged (Beckman Microfuge, 0.1-d tulws') for 1 min. After till-cse st]cc(~ssi~~ lvashings and centrifugations, cells \vere lysetl \vith tlistilletl water ailtl \ver(’ cciltr.ifugetl for two 5-min intervals, discarding pelletetl cell stroma bet\veen celltrifllg;ltions. Hemoglobins were treated with carbon lllouoside ant1 were stcor(L(l at - 70” c until needed. l leinoglobins tvere prepared for polyacrylamitle gel isoelectric focusing ( I’--lGI 1;) l)y coupling an S-ethylamine group to the accessible sulfliytlryl groups, tl7Lls efiectiilg resolution of the structurally different lieiiioglol~i~is (8, 20, 21 ). First, the ~;11111’1(,~ Iverc dihite(l uith dktikd \vater to a ccWXllhXti~Jll Of 20 lllg/lll~. T(J tllis \v;L acl(je[l

414

STEPHENS,

MCIVOR,

AND

WARNER

an equal volume of reducing buffer (0.2 M boric acid, 0.08 M NaOH, 0.1 M EDTA, 0.02 M mercaptoethanol). Modification of the hemoglobin was caused by addition of dry cystamine dihydrochloride (Sigma Chemical Co., St. Louis, MO.) to a final concentration of 0.25 M. Samples were diluted to a final concentration of 5.0 mg/ml with an equal volume of 50% (w/v) sucrose containing 0.04 M potassium cyanide, 0.1 M boric acid, 0.04 M NaOH, 0.005 M EDTA, pH 8.7 (22). The exact concentration of the hemoglobins was verified by the absorption at 576 mn (E = 3.77 X lo4 M-l cm-l) prior to sample application to gels. When artificial mixtures of the structurally different hemoglobins were used, they were prepared before application to the gels. Polyacrylakde gel isoelectric focusing (PAGZF) . The technique of polyacrylamide gel isoelectric focusing was a modification of a previously described method (22, 23) using a Buchler Polyanalyst and a regulated power supply. The gels were prepared as follows. In a 50-d beaker, 8.17 ml of an acrylamide and methylene-bis-acrylamide (Bio-Rad, Richmond, Calif.) stock solution (292.5 g/liter of acrylamide and 14.6 g/liter of methylene-bis-acrylamide) were mixed with 1.40 ml of glycerol, 22.2 ml of distilled water, 0.53 ml of ampholytes (3-10) (BioRad), 0.53 ml of either ampholytes (6-8) (Bio-Rad) or ampholytes (5-S) (LKB) , and 2.24 ml of a solution containing 1% (v/v) TEMED (Bio-Rad) and 0.01% (w/v) riboflavin (Bio-Rad). After mixing, gel tubes (12 X 0.5 cm) were filled within 2 cm of the top, after which water was carefully layered on top of the gel solution. Gels were photopolymerized for 10 hr, and excess water was removed before sample application. Three-hundred and fifty micrograms of artificial mixtures of C57BL/6 and A hemoglobins or allophenic mouse hemoglobins was applied to the surface of the gels. To safeguard against possible denaturation of proteins at the interface by the catholyte, 100 ~1 of 10% sucrose was overlayed on the sample. A current of 1 mA/tube was established until the voltage rose to 500 V. Thereafter, the voltage was maintained at this level for the duration of the experiment. Isoelectric focusing was complete within 4 hr (22, 24). Gels were then removed from their tubes and washed in 10% TCA for 1.5 hr to fix the protein and remove the ampholytes (24). The gels were stored in distilled water until they were scanned, with no apparent deterioration. Hemoglobin quantitation. Gels were scanned at a wavelength of 576 nm using a Beckman ACTA spectrophotometer (Fullerton, Calif.) and a Heath strip chart recorder (Model EU-200-01) (Benton Harbor, Mich.) preset at 10 mV, with a chart speed of 15 set/cm. Total area under the curves was established by tracing with a planimeter. Areas under individual peaks were resolved using the following formula (25) : PI = [hJ(hl + hi + h3)] X (total area), Pz + P3 = total area - PI, where PI = area under largest peak ; P2 = area under intermediate peak ; P3 = area under smallest peak ; lzl = height of largest peak ; hl = height of intermediate peak ; 1~ = height of smallest peak. A standard curve was established by rutlning known mixtures of C57BL/6 and A hemoglobins and drawing the least-squares line through a plot of the area under the C57BL/6 peak versus the known percentage of C57BL/6 hemoglobin which had been applied to the gels. The composition of the hemoglobin of the allophenic

CEIIiVERIC

IJRIFT

IN

ALLOPHENIC

11 5

BII(‘I-

FIG. 1. A plot of the percentage of killing of known mixtures of C57BI,/6 and A peripheral white blood cells (PWBC) with anti-C57BL/6 serum. (-) Theoretical line through the points for a perfect correlation; (---) least-squares line through the points. curve. ;\/lost hemoglobin sanl\vas deterinitled by comparison to the standart pies were run in duplicate, except in a few instances when there xvas sufficient sample for a single run only.

mice

RESULTS

All anti-C57BL/6 serum used showed no significant killing of ,I target cells. Artificial mixtures of C57BL/6 and A cells always gave the predicted killing, as is shown in Fig. 1. All unknown samples were corrected to controls which were included in each determination. This occasionally resulted in values greater than 100%. For instance, if the antiserum killed 94% of the C57BL/6 control cells and 96% of the unknown cells, then the composition of the unknown allophenic 111ouse would be reported as 102% C57BL/6. B ased on many repeated ckter~llillati~J~lS \ve estimate the error in this assay to be from 10 to 209, depending on the strength of tile antiserum used in a particular determinatit~ll.

Figure 2 shows the isoelectric focusing gels of various mixtures of C57BT,/6 aud 21 henmglohins. It is seen that this technique resolves the “diffuse” lieinoglol~in or the A mice into a major hand and a minor hand and that, under our conditions, 110th bands migrate to a point different than the one to which the single band from the C57BL/6 mice migrates. This is a marked advantage of this isoelectric focusing technique compared to other electrophoretic techniques which have lIeen used in the past. It is this selmration of the diffuse (tloul~le) and single lieuwglol)iils that has allowctl us to quaiititate our data. ‘I’lle isoelectric focusing patterns \v(’ cJlJScrvc(l

arc

pdicted

fro111

the

e~Ktr~~hor&c

StlldieS

Of

l~LlttOl1

r’l

tr/.

(26,

27)

416

STEPHENS,

MCIVOR,

AND

WARNER

12 I--

4-

r:098

r

PC0.00 I

r

-. 2 -.

0

x I 10

I 20

I

WBt‘% 30

I

I 50

I 60

I 70

60

1 90

I 100

HEMOGLOBIN

FIG. 3. The llemoglohitl.

area under

tllc C57B12/6 peak as a functim

of the percentage

of C57Rl./i,

that .\ nlice and frtml the sequencing work of Gilnmn (28), which have sho~vn niice l~roprmluce two hemoglobin B chains in uneq~d amounts, while C57HI,/fi tluce a single ,8 chain. Neither A nor C57RI./6 nlice have any know11 electroldmrctic variants in the hemoglol~ili u chain. The gels tvere scanned, and the area under the c‘57131,/6 peak MXS plottrtl a> a function of the percentage of C57BI,/h hemoglol)in on the gel, \vitli the rc.sdts sho~.n in Fig. 3. The cotnposition of the unknown allophenic mice \vas tleternlinctl 1)~ coniparing the area under the C57RT,/6 peak \vitli the standard curve.

Fifteen C.57131J/6 H A allophenic mice were produced for this study. Their coat color comlmsition was estimated visually at the time of neaning. The peripheral \\hite I&xd cell and lienioglol~in compositions were measured at either 3 or 3 months of age (first determination), l-month later (second tleterminatioll), ant1 then 3 nlonths after that (third determination). For all three tleterminations, the peripheral n-hite Mood cell and hemoglobin samples hvere collected within 1 day clF each other. The complete characterization of the allophenic mice is shrmn in ‘l‘alble 1. The change in peripheral white l&.mcl cell coml~ositio~i and lieinoglol~in cornposition of each mouse is plotted as a function of age in Fig. 1. Figure 1~4 includes the five single-colored allophenic mice, n-hereas Figs. 413 aid -I(: include the 10 niulticolorcd allophenic mice. D1SCUSS10S The present study sl~ows that the composition of the peripheral white ldood cells and/or red Mood cells of allophenic mice may change during the course of their lives. For convenience, we will consider significant changes between t\v.z-o tinic points to he greater than or eilual to IOc/c, although, as is seen in Table 1, man!-

418

STEPHENS,

MCIVOR,

AND

WARNER

of the data points are better than the * 20% error in each determination. We have previously termed changes in the chimeric composition of allophenic mice “chimeric drift” (29). In this study, S/1.5 or 33% of the mice (Nos. 158, 162, 173, 187, and 188) showed chimeric drift in their peripheral white blood cell compositions, and 2/15 or 13% of the mice (Nos. 159 and 191) showed chimeric drift in their hemoglobin compositions. Thus, a total of 7/15 or 47% of the mice showed chimeric drift in one or the other of these markers. The mice showing chimeric drift may thus be termed “unstable” chimeras, whereas the mice showing no drift may be termed “stable” chimeras. It is, of course, possible that all allophenic mice would prove to be unstable chimeras if they were examined at more frequent intervals than those reported in this paper. It is seen in Table 1 and Fig. 4 that the degree of internal chimerism of the allophenic mice is independent of their coat color phenotype. Allophenic mice are defined as animals which arise from a double-sized blastocyst (30). This terminology therefore implies nothing about differential cell death or cell proliferation and selection at a later time which might lead to different degrees of chimerism in different tissues. It should be noted that allophenic mice are primary chimeras and, therefore, could be chimeric in any or all of their tissues (5). Cattle twins and marmoset twins, on the other hand, are secondary chimeras and, therefore, are chimeric in the lymphomyeloid system only. Thus, when all of the single-colored animals are examined in Fig. 4A, it is seen that at least two of the all-black animals (Nos. 160 and 187) show evidence of a significant percentage of A-type peripheral white blood cells, and one all-black animal (No. 188) shows evidence of significant A-type hemoglobin. On the other hand, TABLE Analysis Mouse No.

Sex

Percentage of C57BL/6 coat colora

of C57BL/6

0 0 3 3 0 8 0 3 3 0 3 3

167

8

187 188

0 0

0 40 50 50 50 60 60 75 85 9.5 98 100 100 100 100

++ A Allophenic

Percentage First determination PWBC

170 159 162 171 191 169 172 173 168 158 1.57 160

1

4fl 32 f 7 59 f 1 28 f 1 42 f 4 60 f 8 50 f 6 71 f 1 102 f 0 99 f 9 92 f 7 90 f 1 80 f 6 96 f 1 119f5

5f2 49 f 69 f 41 f5 51 f9 76 f 73 76 f

PWBC

6 3

3 4

100

67

of C57BL/6

PWBC

Second determination

Hb

95 96 96 93 80

Mice

3fl 48 f 66f6 32 f 28f0 61 f 46 f 58 f

2

2 18 2

f

11

f

5

44f4 91 f 87 f

Hb

PWBC

6fl 33 f 0 70&O

4fl 14 f 27 f 8fl

1.5 f 3 51 f21

4 4 4

65 f 54 79 f

10 1

99f.5

2 1

106fl

58 f 79 f

Third determination

1

113f7

f f f

1 1

or Hb*

72~1~3 97 f 1 83 f 92f7 9.5 f 93 f

= Coat color was estimated visually at the time of weaning. b The percentage of C57BL/6 peripheral white blood cells (PWBC) was determined as described in the text.

8 12 25

Hb

3 6

11 f3

62 f 36 f 2.5 f0 102 f4 107 f 97f4 57 f 77 f 56 f 84 f

2 0

1

Of0 82 66 f 12 7f2 58 43 f 57 89 f 98 f 89

4

2 3 3

1 1

88 102 f

25

5 6

103 f 93 f

0 18

or of hemoglobin

(Hb)

C:IIIilIEKIC

DRIFT

160

IN

ALLOl’IIE:NIC

167

187 ,A

-- ' \

4

-A

\\

::

/

'

'Z---

I 4

7

12 3

4

/-.

r

62,

\

'\

I 3

410

JlI(‘l<

7

2

3

__

6

AGE of MOUSEbnonths)

162

191

169

yF---

Li-'

;\;

B I !

3

t 6

3

I 7

4

AGE of MOUSE (months) 120

2

158

172

157

100

’

-I2

M;

“,

C L/i _

I

5

1)

4

I 5

8

AGE of MOUSE(monthr) FIG. 4. Hemoglobin and peripheral white blood cell Allophenic mice as a function of ages of mice. (4) (B, C) include the 10 multicolored mice.

(PWBC) Includes

composition of C57BI,/(i i :\ the five single-colored mice;

420

STEPHENS,

MCIVOR,

AND

WARNER

when the 10 multicolored animals are examined in Figs. 4B and 4C, two of them (Nos. 168 and 157) s1low no evidence of the A component in either their peripheral white blood cell or hemoglobin compositions. Likewise, it is seen in Table 1 and Fig. 4 that the degree of internal chimerism is independent of the sex or age of the mice in this study. We have observed that the direction of chimeric drift may be toward either parental cell type or may fluctuate back and forth. In the study of Mintz and Palm (7)) all shifts in the erythrocyte composition of C57BL/6 e C3H allophenic mice were seen in the direction of the C57BL/6 parental type. However, a very limited number of determinations was made, so that it is possible that shifts in the other direction could occur if more mice and more time points were observed. A recent study by West (12) also suggests that there is a temporal shift toward C57BL/6 cells in six out of eight C57BL/6 f) C3H allophenic mice examined. In contrast to these findings is the present study and another study (31) which have shown that chimeric drift in three other combinations of allophenic [ C57BL/6 t) ( AxS JL) Fr ; C57BL/ 6 ti (CBA X CBA/H-T6) F1 ; C57BL/6 * DBA/l] may occur in either parental direction ancl fluctuate back and forth between the two parental cell types. Thus, the particular cell combination in an allophenic animal may determine relative rates of erythropoiesis and whether or not one parental red blood cell type tends to dominate an animal as a function of age. Next, let us examine the correlation of the peripheral white blood cell and hemoglobin compositions of the allophenic mice at each given time point. There is strong evidence that erythrocytes and white blood cells arise from the same pluripotent hematopoietic stem cells during embryogenesis (32). However, the mechanisms for the maintenance of these two cell populations in the adult animals may not be coordinated (8). Figure 5 shows a plot of the hemoglobin composition of the allophenic animals as a function of their peripheral white blood cell composition at any given time point. It is seen that there is a strong statistical correlation

0

20

40

60

60

100

120

%C57BL/6 PWBC FIG. 5. Percentage of C57BL/6 hemoglobin as a function of the percentage C57BL/6 of peripheral white blood cells (PWBC). (-) Theoretical line for a perfect correlation; (---) least-squares line through the points.

CHIMERIC

DRIFT

IN

AILOI’II1~‘iIC

2

‘I‘IIBLE i\nalysis

of Chimrric

Drift

121

31 I(‘I:

in C57BLj6

+* .I .-\llophcnic

5licc.

.-__ Hemoglobin drift

White blood cell drift _----___

White blood cell hemoglobin discordancc~

-~ ~~. l.%159

150 162

162 173 187 188

187 1X8 lY1

a Mouse number.

of the data. However, examination of Table 1 and Fig. 1 shows that four mice (Nos. 159, 162, 187, and 188) show a definite discordance of their peripheral whiti’ blood cell and hemoglobin compositions in at least one point in their lives. Thus, it seems likely that, although the erythrocytes and white blood cells arise from the same embryonic precursor cells, the rate of proliferation of the t\vo cell types in later life is independent. The mechanism of chimeric drift is still unknown. It is interesting to note that all of the animals showing white blood cell-hemoglobin discordance also sho\vetl chimeric drift in either their peripheral white blood cell or hemoglobin compositions at some time in their lives. This fact is summarzed in Table 2. Thus, since drift in the white blood cell and hemoglobin compositions is not coordinate, there does not seem to be one simple mechanism (e.g., a graft versus host reaction) to explain both. Nor does it seem likely that drift in the white blood cell population is the cause of drift in the red cell population. It is possible, however, that there is a11 undetected periodicity to chimeric drift which could be determined bq’ analyzing the mice at closer time intervals. It will be most interesting if future studies shon that the mechanisms of chimeric drift and the mechanisms responsible for the maintenance of the state of tolerance in adult allophenic mice are related to 011~ another. ACKNOWLEDGMENTS This work assistance.

was supported

by NH

Grant

AI

11752. We thank

Ruth

Graves

for

technic;~l

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Tarkowski, A. K., Nature (London) 190, 857, 1961. Mintz, B., Science 138, 594, 1962. Mintz, B., Amer. 2001. 2, 432, 1962. Gardner, R. L., Nature (London) 220, 596, 1968. McLaren, A., “Mammalian Chimaeras.” Cambridge University Press, Cambridge, 1976. Mintz, B., and Palm, J., J. Cell Biol. 27, 66A, 1965. Mintz, B., and Palm, J., J. Exp. Med. 129, 1013, 1969. Wegmann, T. G., and Gilman, C. G., Develop. Biol. 21,281, 1970. Barnes, R. D., Tuffrey, M., Kingman, J., and Risdon, R. A., Clin. Exp. Immunol. 10. 493, 1972.

422

STEPHENS,

MCIV~R,

AND

WARNER

IO. Barnes, R. D., Tuffrey, M., Kingman, J., Thornton, C., and Turner, M. W., Cl&. Exp. Zmmunol. 11, 605, 1972. 11. Barnes, R. D., Tuffrey, M., and Kingman, J., Cli+c. Exp. Zmmzlnol 12, 541, 1972. 12. West, J. D., Exp. Hematol. 5, 1, 1977. 13. Green, E. L. (Ed.), “Biology of the Laboratory Mouse.” McGra\nlHill, New York, 1966. 14. Mintz, B., In “Methods in Mammalian Embryology” (J. C. Daniel, Ed.), pp. 186-214. Freeman, San Francisco, 1971. 15. Warner, C. M., Fitzmaurice, M., Maurer, P. H., Merryman, C. F., and Schmerr, M. J., J. Zmm~nol. 111, 1887, 1973. 16. Batchelor, J. R., In “Handbook of Experimental Immunology” (D. M. Weir, Ed.), pp. 32.132.14, Blackwell Scientific, Oxford, 1973. 17. B?iyum, A., Scund. J. Cl&. Lab. Invest. 21 [Suppl 971, 1, 1968. 18. Gorer, P. A., and O’Gorman, P., Transplant. Bull. 3, 142, 1956. 19. Clegg, M. D., and Schroeder, W. A., J. Amer. Chem. Sot. 81,6065, 1959. 20. Gilman, J. G., Ph.D. Dissertation, University of Wisconsin, Madison, Wis., 1973. 21. Smithies, O., Science 150, 1595, 1965. 22. Righetti, P., and Drysdale, J. W., Biochim. Biophys. Acta 236, 17, 1972. 23. Drysdale, J. W., Righetti, P., and Bunn, H. F., Biochim. Biophys. Acta 229, 42, 1971. 24. Dale, G., and Latner, A. L., Lancet 1, 847, 1968. 25. Instruction Manual Series 200 Disc Integrator. Disc Instruments, Inc., Cosa Mesa, California, 1973. 26. Hutton, J. J., Bishop, J., Schweet, R., and Russell, E. S., Proc. Nat. Aced. Sci. USA 48, 1505, 1962. 27. Hutton, J. J., Bishop, J., Schweet, R., and Russell, E. S. Proc. Nat. Acad. Sci. USA 48, 1718, 1962. 28. Gilman, J. G., Science 178, 873, 1972. 29. Warner, C. M., Graves, R. M., Tollefson, C. M., Schmerr, M. J. F., Stephens, T. J., Merryman, C. F., and Maurer, P. H., Zmmzhnogenetics3, 337, 1976. 30. Mintz, B., Proc. Nat. Acad. Sci. USA 58,344, 1967. 31. Warner, C. M., McIvor, J. L., and Stephens, T. J., Transplantation, in press, 1977. 32. Till, J. E., Price, G. B., Mak, T. W. and McCullock, E. A., Fed. Proc. 34, 2279, 1975.