0 1 A a DT 2 11 shortened shorten VBD 12 16 life life NN 17 21 span span NN 22 24 of of IN 25 32 EKLF-/- eklf-/- JJ 33 38 adult adult JJ 39 51 erythrocytes erythrocyte NNS 51 52 , , , 53 56 due due JJ 57 59 to to TO 60 61 a a DT 62 72 deficiency deficiency NN 73 75 of of IN 76 87 beta-globin beta-globin NN 88 94 chains chain NNS 94 95 , , , 96 98 is be VBZ 99 110 ameliorated ameliorate VBN 111 113 by by IN 114 119 human human JJ 120 132 gamma-globin gamma-globin NN 133 139 chains chain NNS 139 140 . . . 142 147 Using use VBG 148 158 homologous homologous JJ 159 172 recombination recombination NN 172 173 , , , 174 178 both both DT 179 183 EKLF eklf NN 184 191 alleles allele NNS 192 194 in in IN 195 201 murine murine JJ 202 211 embryonic embryonic JJ 212 216 stem stem NN 217 218 ( ( ( 218 220 ES ES NNP 220 221 ) ) ) 222 227 cells cell NNS 228 232 were be VBD 233 244 inactivated inactivate VBN 244 245 . . . 246 251 These these DT 252 259 EKLF-/- eklf-/- JJ 260 262 ES ES NNP 263 268 cells cell NNS 269 273 were be VBD 274 281 capable capable JJ 282 284 of of IN 285 295 undergoing undergo VBG 296 298 in in FW 299 304 vitro vitro FW 305 320 differentiation differentiation NN 321 323 to to TO 324 328 form form VB 329 339 definitive definitive JJ 340 349 erythroid erythroid JJ 350 358 colonies colony NNS 359 363 that that WDT 364 368 were be VBD 369 376 similar similar JJ 377 379 in in IN 380 384 size size NN 385 388 and and CC 389 395 number number NN 396 398 to to TO 399 404 those those DT 405 411 formed form VBN 412 414 by by IN 415 424 wild-type wild-type JJ 425 427 ES es NN 428 433 cells cell NNS 433 434 . . . 435 442 However however RB 442 443 , , , 444 447 the the DT 448 455 EKLF-/- eklf-/- JJ 456 464 colonies colony NNS 465 469 were be VBD 470 476 poorly poorly RB 477 491 hemoglobinized hemoglobinize VBN 492 495 and and CC 496 506 enucleated enucleate VBN 507 519 erythrocytes erythrocyte NNS 520 522 in in IN 523 528 these these DT 529 537 colonies colony NNS 538 547 contained contain VBD 548 556 numerous numerous JJ 557 562 Heinz Heinz NNP 563 569 bodies body NNS 569 570 . . . 571 578 Reverse reverse JJ 579 603 transcriptase-polymerase transcriptase-polymerase NN 604 609 chain chain NN 610 618 reaction reaction NN 619 620 ( ( ( 620 626 RT-PCR RT-PCR NNP 626 627 ) ) ) 628 636 analyses analysis NNS 637 645 revealed reveal VBD 646 650 that that IN 651 656 adult adult JJ 657 660 and and CC 661 670 embryonic embryonic JJ 671 677 globin globin NN 678 683 genes gene NNS 684 688 were be VBD 689 702 appropriately appropriately RB 703 712 regulated regulate VBN 712 713 , , , 714 718 with with IN 719 722 the the DT 723 732 exception exception NN 733 735 of of IN 736 740 beta beta SYM 741 750 h1-globin h1-globin NN 750 751 , , , 752 757 which which WDT 758 767 continued continue VBD 768 770 to to TO 771 773 be be VB 774 783 expressed express VBN 784 786 at at IN 787 788 a a DT 789 793 very very RB 794 797 low low JJ 798 803 level level NN 803 804 . . . 805 808 The the DT 809 814 ratio ratio NN 815 817 of of IN 818 823 adult adult JJ 824 848 beta-globin/alpha-globin beta-globin/alpha-globin NN 849 853 mRNA mrna NN 854 856 in in IN 857 860 the the DT 861 867 mutant mutant JJ 868 870 ES ES NNP 871 876 cells cell NNS 877 880 was be VBD 881 885 1/15 1/15 CD 886 888 of of IN 889 893 that that DT 894 896 in in IN 897 906 wild-type wild-type JJ 907 909 ES es NN 910 915 cells cell NNS 915 916 . . . 917 921 When when WRB 922 925 the the DT 926 933 EKLF-/- eklf-/- JJ 934 939 cells cell NNS 940 944 were be VBD 945 953 injected inject VBN 954 958 into into IN 959 970 blastocysts blastocyst NNS 970 971 , , , 972 976 they they PRP 977 980 did do VBD 981 984 not not RB 985 995 contribute contribute VB 996 998 at at IN 999 1000 a a DT 1001 1011 detectable detectable JJ 1012 1017 level level NN 1018 1020 to to TO 1021 1024 the the DT 1025 1031 mature mature JJ 1032 1043 erythrocyte erythrocyte NN 1044 1055 compartment compartment NN 1056 1058 of of IN 1059 1062 the the DT 1063 1071 chimeric chimeric JJ 1072 1079 animals animal NNS 1079 1080 , , , 1081 1086 based base VBN 1087 1089 on on IN 1090 1098 analysis analysis NN 1099 1101 of of IN 1102 1109 glucose glucose NN 1110 1119 phosphate phosphate NN 1120 1131 isomerase-1 isomerase-1 NN 1132 1133 ( ( ( 1133 1138 GPI-1 gpi-1 NN 1138 1139 ) ) ) 1140 1148 isozymes isozyme NNS 1149 1152 and and CC 1153 1164 hemoglobins hemoglobin NNS 1165 1169 that that WDT 1170 1181 distinguish distinguish VBP 1182 1184 ES es NN 1185 1197 cell-derived cell-derived JJ 1198 1210 erythrocytes erythrocyte NNS 1211 1215 from from IN 1216 1220 host host NN 1221 1239 blastocyst-derived blastocyst-derived JJ 1240 1252 erythrocytes erythrocyte NNS 1252 1253 . . . 1254 1256 In in IN 1257 1265 contrast contrast NN 1265 1266 , , , 1267 1283 semiquantitative semiquantitative JJ 1284 1290 RT-PCR RT-PCR NNP 1291 1299 analysis analysis NN 1300 1302 of of IN 1303 1306 RNA RNA NNP 1307 1311 from from IN 1312 1325 reticulocytes reticulocyte NNS 1326 1328 of of IN 1329 1332 the the DT 1333 1337 same same JJ 1338 1346 chimeric chimeric JJ 1347 1354 animals animal NNS 1355 1364 suggested suggest VBD 1365 1369 that that IN 1370 1373 the the DT 1374 1376 ES ES NNP 1377 1389 cell-derived cell-derived JJ 1390 1403 reticulocytes reticulocyte NNS 1404 1408 were be VBD 1409 1416 present present JJ 1417 1419 at at IN 1420 1421 a a DT 1422 1427 level level NN 1428 1430 of of IN 1431 1432 6 6 CD 1432 1433 % % NN 1434 1436 to to TO 1437 1438 8 8 CD 1438 1439 % % NN 1439 1440 . . . 1441 1445 This this DT 1446 1455 indicated indicate VBD 1456 1460 that that IN 1461 1464 the the DT 1465 1472 EKLF-/- eklf-/- JJ 1473 1485 erythrocytes erythrocyte NNS 1486 1488 in in IN 1489 1494 adult adult JJ 1495 1502 animals animal NNS 1503 1507 must must MD 1508 1510 be be VB 1511 1522 short-lived short-lived JJ 1522 1523 , , , 1524 1534 apparently apparently RB 1535 1538 due due JJ 1539 1541 to to TO 1542 1545 the the DT 1546 1555 imbalance imbalance NN 1556 1558 of of IN 1559 1564 beta- beta- NN 1565 1571 versus versus CC 1572 1584 alpha-globin alpha-globin NN 1585 1591 chains chain NNS 1591 1592 , , , 1593 1600 leading lead VBG 1601 1603 to to TO 1604 1607 the the DT 1608 1621 precipitation precipitation NN 1622 1624 of of IN 1625 1631 excess excess JJ 1632 1644 alpha-globin alpha-globin NN 1645 1651 chains chain NNS 1652 1654 to to TO 1655 1659 form form VB 1660 1665 Heinz Heinz NNP 1666 1672 bodies body NNS 1672 1673 . . . 1674 1684 Consistent consistent JJ 1685 1689 with with IN 1690 1694 this this DT 1695 1705 hypothesis hypothesis NN 1705 1706 , , , 1707 1710 the the DT 1711 1716 short short JJ 1717 1721 life life NN 1722 1726 span span NN 1727 1730 was be VBD 1731 1742 ameliorated ameliorate VBN 1743 1745 by by IN 1746 1758 introduction introduction NN 1759 1763 into into IN 1764 1767 the the DT 1768 1775 EKLF-/- eklf-/- JJ 1776 1778 ES ES NNP 1779 1784 cells cell NNS 1785 1787 of of IN 1788 1789 a a DT 1790 1795 human human JJ 1796 1812 LCR/gamma-globin LCR/gamma-globin NNP 1813 1817 gene gene NN 1817 1818 , , , 1819 1821 as as IN 1822 1831 evidenced evidence VBN 1832 1834 by by IN 1835 1838 the the DT 1839 1847 presence presence NN 1848 1850 of of IN 1851 1853 ES ES NNP 1854 1866 cell-derived cell-derived JJ 1867 1880 reticulocytes reticulocyte NNS 1881 1883 as as RB 1884 1888 well well RB 1889 1891 as as IN 1892 1898 mature mature JJ 1899 1911 erythrocytes erythrocyte NNS 1912 1914 in in IN 1915 1918 the the DT 1919 1924 blood blood NN 1925 1927 of of IN 1928 1931 the the DT 1932 1940 chimeric chimeric JJ 1941 1948 animals animal NNS 1948 1949 . . .