Maternal effect on GFP expression. (A) Mating schemes of heterozygous (+/–)F₁ and F₂ adults with WT (–/–) zebrafish. F₂ and F3 embryos were scored at the 1,000-cell stage. In F₂ adults, the LTR-GFP transgene could be derived from either the mother or the father as the straight and diagonal arrows indicate. The results were compiled from analyses of 20–100 F₂ and F₃ embryos of Tg lines 1–4. (B) Microscopic and PCR analyses of F₂ embryos. (Left) F₂ embryos from a heterozygous F₁ female mated with WT male. (Right) F₂ embryos from a heterozygous F₁ male mated with WT female. (Lower) The PCR bands amplified from DNAs isolated from individual F₂ embryos at the 1,000-cell stage. PCR primer pair spanned 650 bp of transgene DNA. First lanes, the 100-bp markers. F₂ embryos of lines 1 and 4 were analyzed by PCR and produced the same results; the results of line 1 are shown.

In situ hybridization of Tg zebrafish. (A) Ovary section stained with hematoxylin/eosin dyes. O, oocytes; M, muscle cells. (B) Ovary section hybridized with the antisense GFP probe. Oocytes of different sizes contain positively stained blue cytoplasm surrounding the unstained nuclei. (C) Boxed area in B viewed under a higher magnification. Large cell at the top, ≈1.5 mm in diameter, is the size of a mature oocyte; adjacent smaller cells with blue cytoplasm are immature oocytes or nurse cells. (D) Ovary section hybridized with the sense GFP probe. (E and F) Testis sections stained with hematoxylin/eosin dyes and viewed under a higher magnification. S, spermatozoa, stained dark purple by the dyes, within the seminiferous tubule enclosed by the unstained basement membrane. (G) Testis hybridized with the antisense probe. S, spermatozoa, which did not produce hybridization signals and appeared unstained within the seminiferous tubule. (H) Testis hybridized with the sense probe. The spermatozoa within the seminiferous tubules did not produce hybridization signals. (I and J) Sections of intestine (Int.), kidney (K), and muscle (M) hybridized to the antisense and sense probes, respectively. (I Inset) Enlarged view of the intestinal microvilli and crypts with positive blue staining; the crypts at the bases of the microvilli are underneath the unstained submucosa at the top of Inset. To save figure space, the kidney was moved from its location near the swim bladder. (K and L) Sections of scales and dorsal fin hybridized to the antisense and sense probes, respectively. Sections shown were made from adult fish of line 1; adult female and male fish of lines 3 and 4 produced the same hybridization results (not shown).

Transcriptional status of the β-globin gene locus analyzed by RT-PCR. (A) Map of the β-globin gene locus. Angled arrows, transcriptional direction of the ERV-9 LTR, the β-LCR, and the β-like globin genes determined by directional RT-PCR (6) in CD34⁺ cells and erythroblasts (data not shown). Horizontal lines labeled 1–7, RT-PCR products generated by primer pairs 1–7; numbers below, sizes in bp of the RT-PCR products; lines 4–6 and bent lines underneath, RT-PCR products generated, respectively, from unspliced and spliced globin RNAs. Other designations are the same as in Fig. 1 A. (B) Lanes 1–7, RT-PCR products generated, respectively, by primer pairs 1–7 from various human tissues and cells resolved in agarose gels. Lanes 8, RT-PCR band generated from β-actin mRNA as internal quantitative control. Lanes M, 100-bp size marker ladder with the 500-bp band marked. In lanes 1 of the blood and keratinocyte panels, the larger band of 600 bp was a nonspecific RT-PCR product containing short segments of ERV-9 LTR and L1 DNAs partially aligned with >200 GenBank files.

Generation of Tg zebrafish. (A) The humanβ-globin gene locus. Hatched box, the ERV-9 LTR; solid boxes, hypersensitive sites HS5, 4, 3, 2, and 1 defining theβ-LCR; striped boxes, the embryonic ε-, the fetal γ-, and the adult δ- and β-globin genes. In the enlarged map of the ERV-9 LTR: box containing 14 arrowheads, the U3 enhancer (E) containing 14 tandem repeats, each of 40 bp; P and R, the LTR promoter and R regions; box with three horizontal arrows, the U5 region with three 80-bp tandem repeats. Angled arrow, initiation site of sense transcription activated by the LTR enhancer; stippled box, the R-U5 region probe for in situ hybridization with human tissues (Fig. 5). (B) Map of the LTR-GFP transgene plasmid. Bent line, the simian virus 40 intron. Ase and Ssp, AseI and SspI sites to excise LTR-GFP DNA for microinjection; H, HindIII site used to cleave Tg zebrafish DNA for Southern blots; dotted lines, zebrafish DNA flanking the integrated transgene; bracket of 1,660 bp, the minimum size of HindIII bands spanning the transgene integration site; vertically striped box, probe for Southern blots (C) and in situ hybridization with Tg zebrafish (Fig. 4). (C) Southern blots of Tg lines 1–4 (lanes 1–4, respectively), cleaved with HindIII. M, 1-kb size marker ladder.

Expression of the maternal LTR-GFP transgene in Tg embryos. Fluorescent microscope images are shown. (A) Oocyte. (B) One-cell embryo. (C) Two-cell embryo. (D) Eight-cell stage, 1 hpf, side view. (E) Sixteen-cell stage, 1.5 hpf, anterior view. (F) 1,000-cell stage, 3 hpf, side view. (G) Shield stage, 6 hpf. (H) Tail bud stage, 10 hpf, side view. (I) Fifteen-somite stage, 16 hpf. (J) Twenty-four-hpf embryo. (K) Forty-eight-hpf Tg zebrafish. (L) Forty-eight-hpf wt zebrafish. Embryos produced by females of lines 1–4 showed the same pattern of GFP expression. Line 1 embryos are shown.

In situ hybridization of human tissues. (A) Thin section of a 12-year-old ovary hybridized with antisense R-U5 probe. Positive hybridization signals appear as black spots. (B) Higher magnification of boxed area in A. The arrow marks a secondary follicle containing an eccentrically located oocyte; primordial and primary follicles of smaller sizes are detected also in the cortex. (C) Ovary section hybridized with sense R-U5 probe. (D) Higher magnification of boxed area in C. (E) Thin section of a 5-month-old testis hybridized with antisense R-U5 probe. Vertical bar, the external fibrous tunica albuginea capsule. A 39-year-old testis produced similar results (not shown). (F and G) Higher magnifications of E. Outline of a single seminiferous tubule is indicated; arrows mark the Sertoli cells. (H) Testis section hybridized with sense R-U5 probe. (I and J) Sections of the cranial bone of a 1-year-old boy hybridized to antisense and sense probes. Bone marrow space is marked by asterisks; arrows mark the endosteum containing osteoprogenitor cells next to the marrow space. (M and N) Enlarged views of the endosteum in I and J, respectively. Arrows mark positive black staining in areas containing the osteoblasts but not in deeper areas within the mineralized bone containing the differentiated osteocytes (blue cells). (K and L) Skin from a 20-year-old man hybridized with antisense and sense probes. Arrows mark the basal germinal cells between the epidermis (E) and the dermis (D). (O and P) Small intestine hybridized to the antisense and sense probes, respectively. Signals were detected in the crypts at the base of the microvilli, marked by arrows, but not in the submucosa and the intestinal muscle wall to the right of the crypts.