Adult mice cloned from migrating primordial germ cells.
We previously reported that the genomes of gonadal germ cells at 11.5-19.5 days postcoitum (dpc) are incompetent to support full-term development of cloned mouse embryos. In this study, we performed nuclear transfer using primordial germ cells (PGCs) from earlier stages at 8.5-10.5 dpc. When PGC nuclei at 8.5, 9.5, and 10.5 dpc were transferred into enucleated oocytes, seven cloned embryos developed into full-term offspring. Of these, five, all derived from 8.5- or 9.5-dpc PGCs, developed into healthy adults with normal fertility. Of the remaining two offspring derived from 10.5-dpc PGCs, one died shortly after birth, and the other showed slight growth retardation but subsequently developed into a fertile adult. We examined allele-specific methylation at the imprinted H19 and Snrpn loci in 9.5- to 11.5-dpc PGCs. Although the beginning of methylation erasure was evident on the H19 paternal allele at 9.5 dpc, most PGCs did not demonstrate significant erasure of paternal allele-specific methylation until 10.5 dpc. Maternal allele-specific methylation was largely erased from Snrpn by 10.5 dpc. By 11.5 dpc, the majority of PGCs showed nearly complete or complete erasure of allele-specific methylation in both H19 and Snrpn. These results demonstrate that at least some genomic imprints remain largely intact in 8.5- to 9.5-dpc PGCs and then undergo erasure at approximately 10.5 dpc as the PGCs enter the genital ridges. Thus, migrating PGCs at 8.5-9.5 dpc can be successfully used as donors for nuclear transfer, whereas gonadal PGCs at 11.5 dpc and later are incompetent to support full-term development.
Yamazaki
Yukiko
Y
Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96822, USA. yyamazak@hawaii.edu
Low
Eleanor W
EW
Marikawa
Yusuke
Y
Iwahashi
Kazuhiro
K
Bartolomei
Marisa S
MS
McCarrey
John R
JR
Yanagimachi
Ryuzo
R
D000367:Age Factors; D000469:Alkaline Phosphatase; D000818:Animals; D001324:Autoantigens; D019976:Cloning, Organism / Q000379:methods*; D019175:DNA Methylation; D017931:DNA Primers; D004624:Embryo Transfer; D018392:Genomic Imprinting / Q000502:physiology*; D005854:Germ Cells / Q000166:cytology* / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D053652:Nuclear Transfer Techniques; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013447:Sulfites; D055517:snRNP Core Proteins
D001324:Autoantigens; D017931:DNA Primers; C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D017411:Ribonucleoproteins, Small Nuclear; D013447:Sulfites; D055517:snRNP Core Proteins; D000469:Alkaline Phosphatase; C042345:hydrogen sulfite