Germ cell expression of an isolated human endogenous retroviral long terminal repeat of the HERV-K/HTDV family in transgenic mice - PubMed (original) (raw)

Germ cell expression of an isolated human endogenous retroviral long terminal repeat of the HERV-K/HTDV family in transgenic mice

A E Casau et al. J Virol. 1999 Dec.

Abstract

In contrast to most other human endogenous retroviral families, various HERV-K members have open reading frames that code for functional viral proteins which can form noninfectious particles in some germ cell tumors. The HERV-K viral genes are highly transcribed in germ cell tumors but are transcribed to lower or undetectable levels in most other tissue and tumor types. To further analyze the expression patterns of these proviruses, long terminal repeats (LTRs) were isolated from the human genome and used in reporter gene assays. Expression of some HERV-K LTRs was found to be high in human and murine germ cell tumors (testicular teratocarcinomas) and low in non-germ-cell tumors. Furthermore, upon differentiation of a teratocarcinoma cell line, the expression of an active LTR dropped dramatically, suggesting developmental regulation of these proviral LTRs. Transgenic mice harboring an active LTR driving lacZ expression were generated and analyzed. Adult mouse testes showed the highest levels of expression, and the transgene staining appeared to be restricted primarily to the more undifferentiated spermatocytes. Most other tissues analyzed revealed very low or undetectable levels of expression both by reverse transcription-PCR and by Northern blot analysis. Whether the restricted expression of HERV-K in germ cells and in germ cell-derived tumors is of significant importance during development or tumorigenesis remains to be elucidated. Germ line expression of these viruses would allow for their expansion and movement, while somatic repression would ensure limited insertional mutagenesis and misexpression in an individual.

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Figures

FIG. 1

Nucleotide sequence homology between HERV-K LTRE3 (KE3: nt 1 to 921) and HERV-K10 5′ LTR (K10: nt 78 to 999). Identities are depicted as dots. The TATA box and polyadenylation signal are capitalized. The potential glucocorticoid/enhancer site is underlined.

FIG. 2

LTRE3 expression of a reporter gene is high in GCT cell lines (A) and low in non-GCT cell lines (B). The HERV-K LTRE3 was cloned upstream of the luciferase reporter gene and was transiently transfected into various cell lines along with a β-gal control vector to normalize for transfection efficiency. The relative luciferase level value is the light unit ratio between the luciferase vector (Pro-Luc) with promoter alone (SV40 early promoter) and the luciferase vector with promoter and LTRE3 (LTR-Pro-Luc).

FIG. 3

LTRE3 expression is downregulated upon differentiation of an EC cell line. (A) The HERV-K LTRE3 was cloned upstream of a lacZ reporter gene in a vector devoid of any promoter sequences and transiently transfected into the murine EC cell line, F9, along with a luciferase control vector to normalize for transfection efficiency. The relative lacZ level value is the ratio of the promoterless lacZ vector with that of the other vectors: no lacZ (no DNA as a negative control), LTR-lacZ (LTRE3-lacZ), and SV40 lacZ (lacZ vector with the SV40 promoter as a positive control). (B) F9 cells were also stably transfected with the LTRE3-lacZ vector and a neo vector to select for G418-resistant colonies. Colonies were pooled and stained with X-Gal to look for lacZ staining. Panel 1 shows the F9 cells stained with X-Gal. Panel 3 shows the same field by phase-contrast microscopy to show cell morphology. This pooled stable F9 cell line was also differentiated for 1 week with 10−6M RA. Panel 2 shows the differentiated F9 cells stained with X-Gal. Panel 4 shows the same field by phase-contrast microscopy to show the differentiated cell morphologies.

FIG. 4

Two independent transgenic lines express the lacZ gene in adult testes, but at different levels. (A) RT-PCR analysis followed by Southern blotting reveals that only lines Tg7 and Tg9 express the reporter gene. R+ refers to the ROSAβ-geo26 lacZ line, S− refers to the nontransgenic control line, while Tg2, Tg4, Tg7, and Tg9 refer to the transgenic lines. Testis RNA was DNase treated and reverse transcribed in the +RT gel (but not in the −RT gel) and PCR amplified with lacZ primers. (B) RNA integrity was checked by using the RT reactions to PCR amplify a ubiquitous gene, tpi. (C) Total testis RNA was used for Northern blot analysis and hybridized with a lacZ probe. (D) mRNA was isolated for Tg7 and Tg9 and hybridized to a lacZ probe.

FIG. 5

RT-PCR analysis of tissue RNA from transgenic mice reveals the strongest expression in adult testes and, to a lesser extent, in adult brain. Tissues selected from the two expressing transgenic lines Tg7 and Tg9 were Br (brain), Ht (heart), Ts (testis), It (intestines), Kd (kidney), Lg (lung), Lv (liver), Sk (skeletal muscle), Sp (spleen), Th (thymus), Ut (uterus), and Ov (ovary). Controls included negative control tissues from 129Sv nontransgenic mice (S−) and positive control tissues from a constitutive _lacZ_-expressing ROSAβ-geo26 strain (R+). All isolated RNAs were DNase treated for 30 min, and 1 μg was used for each RT-PCR. The first row (+RT β-gal) is RNA that was reverse transcribed with dT primers and PCR amplified with β-gal primers. The second row (−RT β-gal) is RNA that was not reverse transcribed but was PCR amplified with β-gal primers. The third row (+RT Tpi) is the same reverse-transcribed RNA as in the first row but PCR amplified with tpi primers (a ubiquitously expressed gene).

FIG. 6

Immunofluorescence studies reveal staining in the more undifferentiated spermatocytes of the testes of transgenic line Tg7. Frozen sections of adult testes were incubated with a polyclonal antibody against β-gal to detect lacZ staining. (A) ROSA+ control with primary β-gal antibody and secondary Texas Red antibody. (B) ROSA+ with only primary β-gal antibody. (C) ROSA+ with only secondary Texas Red antibody. (D to F) Three sections of nontransgenic testes stained with β-gal and Texas Red antibodies. (G to I) Three sections of transgenic line Tg7 stained with β-gal and Texas Red antibodies. ROSA+ shows strongest staining in the more differentiated cell types of the seminiferous tubules (found near the lumen of the seminiferous tubules), while the negative control 129Sv has a faint amount of background. Sections of testes from Tg7 exhibit staining which is restricted primarily to the more undifferentiated spermatocytes found on the periphery of the seminiferous tubules.

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Germ cell expression of an isolated human endogenous retroviral long terminal repeat of the HERV-K/HTDV family in transgenic mice - PubMed (original) (raw)