Infection Of Preimplantation Embryos Research Articles

Structure and Expression of a Gene Encoding a Putative GTP-Binding Protein Identified by Provirus Integration in a Transgenic Mouse Strain

The Mov-10 mouse strain was derived by infection of preimplantation embryos with the Moloney murine leukemia virus and carries one copy of the provirus in its germ line. Here we show that the provirus has integrated into an evolutionarily conserved gene that can code for a protein of 110 kDa containing the three consensus elements characteristic for GTP-binding proteins. The Mov-10 locus was expressed in a variety of cell types, including embryonal carcinoma and embryonic stem cells. Transcription of the gene was down-regulated about 10-fold when F9 embryonal carcinoma cells are differentiated into parietal endodermlike cells and about 2-fold when they are differentiated into visceral endodermlike cells. High levels of Mov-10 transcripts were also found at different stages of embryonal development and in the testes and thymus of adult animals. Expression was cell cycle controlled, with steady-state RNA levels significantly higher in growth-arrested than in growth-stimulated cells. The results suggest that the Mov-10 locus has an important function in development and/or control of cell proliferation. The provirus was shown to have integrated into intron 1 of the gene without disrupting expression, indicating that integration into intronic sequences of a transcription unit does not necessarily affect transcription. This result together with previous results from the Mov-13 mouse strain suggested that proviruses exert their mutagenic effect only by integration in specific sites, such as cis-regulatory DNA elements.

Structure and expression of a gene encoding a putative GTP-binding protein identified by provirus integration in a transgenic mouse strain.

The Mov-10 mouse strain was derived by infection of preimplantation embryos with the Moloney murine leukemia virus and carries one copy of the provirus in its germ line. Here we show that the provirus has integrated into an evolutionarily conserved gene that can code for a protein of 110 kDa containing the three consensus elements characteristic for GTP-binding proteins. The Mov-10 locus was expressed in a variety of cell types, including embryonal carcinoma and embryonic stem cells. Transcription of the gene was down-regulated about 10-fold when F9 embryonal carcinoma cells are differentiated into parietal endodermlike cells and about 2-fold when they are differentiated into visceral endodermlike cells. High levels of Mov-10 transcripts were also found at different stages of embryonal development and in the testes and thymus of adult animals. Expression was cell cycle controlled, with steady-state RNA levels significantly higher in growth-arrested than in growth-stimulated cells. The results suggest that the Mov-10 locus has an important function in development and/or control of cell proliferation. The provirus was shown to have integrated into intron 1 of the gene without disrupting expression, indicating that integration into intronic sequences of a transcription unit does not necessarily affect transcription. This result together with previous results from the Mov-13 mouse strain suggested that proviruses exert their mutagenic effect only by integration in specific sites, such as cis-regulatory DNA elements.

Tissue-specific and ectopic expression of genes introduced into transgenic mice by retroviruses.

Recombinant retroviruses containing the complete genomic human beta globin gene (under the control of its own promoter) and the bacterial neomycin phosphotransferase gene (under the control of the normal or enhancerless viral promoter) were used to derive transgenic mouse strains by infection of preimplantation embryos. Expression of the beta globin gene in hematopoietic tissues was observed in all transgenic strains. In addition, one strain showed ectopic expression of beta globin in the same tissues that also expressed high levels of RNA from the viral promoter. It is likely that expression from the long terminal repeat (LTR), in contrast to expression from the internal promoter, is dependent on the site of integration. Thus, retroviral vectors can be used for tissue-specific expression of foreign genes in transgenic mice, as well as for the identification of loci that allow developmental activation of a provirus.

Insertion of a bacterial gene into the mouse germ line using an infectious retrovirus vector.

Using a Moloney leukemia virus vector containing the bacterial neo gene, we demonstrate that retrovirus vectors can be used to introduce genes into the mouse germ line. Infection of preimplantation embryos with the vector MLV-NEO.1 resulted in integration of neo sequences in approximately equal to 10% of the progeny mice. One of these animals, mouse F.2, contained approximately six MLV-NEO.1 proviruses at independent integration sites, each present at less than a single copy per cell. This mosaic mouse transmitted one of these proviruses to her offspring, producing a line of transgenic mice carrying a full-length, unrearranged MLV.NEO.1 provirus at a single chromosomal integration site. Mice homozygous at this MLV-NEO.1 locus have also been produced. No expression of the neo gene has been detected in the transgenic mice, either by screening of primary bone marrow or lung cells for resistance to G418 or by RNA transfer blot analysis of RNA from several tissues. In addition, the neo gene was found to be extensively methylated in the transgenic mice; however, treatment of primary cells with 5-azacytidine did not induce G418 resistance. The inactivity of the MLV-NEO.1 provirus in transgenic mice and potential means of eliciting neo expression under these conditions are discussed.

Insertion of the bacterial gpt gene into the germ line of mice by retroviral infection.

Mouse substrains genetically transmitting the exogenous Moloney murine leukemia virus (Mo-MuLV) at a single locus have been derived previously by infection of preimplantation embryos. Here we explore the potential of retroviral vectors for transferring nonviral genes into the germ line of mice. Preimplantation mouse embryos were cocultivated with a cell line that produces a recombinant retrovirus whose genome carries the Escherichia coli gene gpt. We show that the vector sequence was inserted into the genome of the embryo and into the germ line at a frequency similar to that for the Mo-MuLV-helper sequence. A new mouse strain, Mgpt-1, was developed that is homozygous for a single MSVgpt proviral genome. The proviral sequences were highly methylated and not expressed in tissues of Mgpt-1 mice. When cells derived from transgeneic animals were treated with 5-azacytidine, the proviral sequences were not methylated and were transcriptionally activated. These results indicate that nonviral genes that are under the control of the viral long terminal repeat are inactivated when transferred into the germ line of animals.

Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus.

Mice were infected with the exogenous Moloney leukemia virus (M-MuLV) at two different stages of development. Either newborn mice (which can be considered as essentially fully differentiated animals) or preimplantation mouse embryos (at the 4-8 cell stage) were infected with M-MuLV. In both cases, animals that had developed an M-MuLV-induced leukemia were obtained. Two lines of evidence indicate that infection of preimplantation embryos, in contrast to infection of newborns, can lead to integration of the virus into the germ line. 1. Viremic males of the first backcross generation (N-1 generation) transmitted the virus to 50% of their offspring (N-2 generation) when mated with uninfected females. Likewise, a 50% transmission was observed from viremic N-2 and N-3 males to the next generations. 2. Molecular hybridization experiments revealed that viremic N-1 and N-2 animals carried one copy of M-MuLV per diploid mouse genome equivalent in all "non-target" organs tested. Together, both experiments indicate that the exogenous M-MuLV can be converted to an endogenous virus after infection of preimplantation embryos. The available evidence suggests that M-MuLV integrated into the germ line at one out of two possible integration sites. Thus, viremic backcross animals are heterozygous for a single Mendelian locus carrying the M-MuLV gene. During leukemogenesis an amplification of the M-MuLV from one copy to a maximum of four copies per diploid mouse genome equivalent takes place in the tumor tissues.