Specific expression of the urotensin II gene in sacral motoneurons of developing rat spinal cord.

The neuropeptide urotensin II (UII) is expressed in motoneurons of the brainstem and spinal cord in adults. Here, the expression pattern of the UII gene was studied in the developing rat spinal cord. UII mRNA was detected by reverse-transcription-polymerase chain reaction (RT-PCR) as early as E10. From E14 to E21, in situ hybridization revealed intense expression of the UII gene specifically in sacral motoneurons, while only faint expression was detected at cervical and thoracic levels. After birth (P0, P4), the expression of UII mRNA increased in motoneurons at all rostrocaudal levels. Thus, UII is the first gene reported to show expression limited to the sacral pool of motoneurons, which are known to have particular properties in terms of targets and programmed cell death.

Cre/loxP-mediated excision of a neomycin resistance expression unit from an integrated retroviral vector increases long terminal repeat-driven transcription in human hematopoietic cells.

Recombinant retroviruses are currently the most attractive vehicles for gene transfer into hematopoietic cells. Retroviral vectors often contain an easily selectable marker gene in addition to the gene of interest. However, the presence and selection for expression of the selectable gene often result in a significant reduction of the expression of the gene of interest in the transduced cells. In order to circumvent this problem, we have developed a Cre/loxP recombination system for specific excision of the selectable expression unit from integrated retroviruses. A retroviral vector, containing both a neomycin resistance expression unit flanked by loxP sites and granulocyte-macrophage colony-stimulating factor cDNA, was used to transduce the human hematopoietic K-562 cell line. Four transduced cell clones were then superinfected with a retrovirus containing a Cre recombinase expression unit. Molecular analyses of 30 doubly transduced subclones showed a strict correlation between cre expression and loxP-flanked selectable cassette excision, thus implying that Cre recombinase activity is very efficient in a retroviral context. Moreover, the excision of the selectable cassette results in a significant increase of granulocyte-macrophage colony-stimulating factor transcription driven by the retroviral promoter.

The V(D)J recombinational and transcriptional activities of the immunoglobulin heavy-chain intronic enhancer can be mediated through distinct protein-binding sites in a transgenic substrate.

Immunoglobulin and T-cell receptor gene transcriptional enhancers encompass sequences which stimulate V(D)J recombination of associated variable gene segments. To address the question of whether enhancer-mediated transcriptional activation and recombinational activation depend on the same cis-regulatory sequences, we have produced transgenic mice by using recombination substrates containing various mutations in the immunoglobulin heavy-chain intronic enhancer (E mu). Analysis of substrate rearrangements indicated that specific compound elements including E-box transcriptional motifs are crucial for the recombinational activity of E mu in the developing B and T lymphocytes. In most cases, a faithful correlation between the levels of substrate germ line transcription and recombination was observed. However, some of the E mu mutants which were able to activate transcription of the unrearranged substrate were inefficient in stimulating transgene recombination, implying that the latter function depends on molecular events other than the mere activation of transcription and that both activities can be mediated through distinct regulatory sequences. Together, these results support a model in which lymphoid gene enhancers, in addition to providing docking sites for factors that dictate transcriptional accessibility, must have some specific function(s) for activating V(D)J recombination.

Sequences affecting the V(D)J recombinational activity of the IgH intronic enhancer in a transgenic substrate.

The immunoglobulin heavy chain intronic transcriptional enhancer (E mu) is part of a complex cis-regulatory DNA region which has notably been shown to modulate V(D)J rearrangements of associated variable gene segments. We have used recombination substrates comprised of the E mu enhancer together with various lengths of additional downstream mu sequences to assess the individual contribution of those sequences to the V(D)J recombinational regulatory activity. Surprisingly, in the absence of large amounts of mu sequences, substrate rearrangements were not detected in Southern blot analyses of the lymphoid tissues from independent transgenic mice, but were readily detectable following transfection into cultured pre-B cells. A short mu segment which includes matrix association regions (MARs) was not sufficient to restore high levels of rearrangements within the reporter transgenes. However, additional experiments demonstrated that the mu sequences are dispensable for V(D)J recombination in transgenic thymuses, implying a suppressive effect exerted by the vector sequences left in the transgenic insert, when they are attached near the E mu regulatory region. This suppression of V(D)J recombination, which correlates with an hypermethylation of the transgenes, is discussed in view of previously reported transgenic and gene targeting experiments.

TCR beta and TCR alpha gene enhancers confer tissue- and stage-specificity on V(D)J recombination events.

We describe transgenic mice carrying germline variable gene segments associated with either the T cell receptor (TCR) beta or alpha gene enhancers (E beta or E alpha). Transgenic constructs underwent high rates of site-specific rearrangements predominantly in T cells from independent mice. Rearrangements of the E beta-containing transgenes began at different stages of T cell differentiation in embryonic and adult thymus than did the E alpha-containing ones, with a pattern superimposable upon the patterns of TCR beta or TCR alpha gene expression, respectively. We demonstrate that sequences within the TCR beta and TCR alpha gene enhancers confer tissue- and stage-specificity upon the V(D)J recombination events affecting adjacent gene segments. The patterns of transgene expression also gave information on developmental events and lineage relationships (gamma delta versus alpha beta) during T cell development.