# VIRAL GENE EXPRESSION IN EMBRYONAL CARCINOMA CELLS

> **NIH NIH R01** · JACKSON LABORATORY · 1987 · $137,424

## Abstract

The objectives of this proposal are focussed on gene regulation in murine
embryonal carcinoma (EC) cells as a model system using recombinant viral
genes as molecular probes.  EC cells are equivalent to the embryonic stem
cells and viral genes are useful probes because undifferentiated EC cells
cannot express the genes of certain viruses (e.g. retroviruses) but they
can do so once differentiated.  To understand the molecular mechanisms of
the block to the viral gene expression, novel EC cell clones were
isolated:  By infection of EC cells with a recombinant transducing
retrovirus that carries the neomycin resistance (neo) gene linked to the
Moloney murine leukemia virus (M-MuLV) LTR, transductant EC clones that
grew in the presence of a neomycin analogue, G418 were isolated.  These
transductant clonal lines are as undifferentiated as the parental EC cells
according to the criteria tested.

The expression of the neo gene of the recombinant provirus can be explained
by cis-acting mechanisms.  These provirus genomes have been molecularly
cloned together with the flanking cellular sequences.  Many of these lambda
clones transfected parental EC cell lines at much higher efficiency than
control plasmid with LTR-neo sequence.  Two possible mechanisms are
suggested:  (a) the provirus genome is integrated in a vicinity of a
cellular enhancer element, and/or (b) the provirus genome has obtained
mutation/rearrangement in LTR regulatory region.  To test these
possibilities, the 5' flanking cellular sequence has been isolated from one
of such clones and inserted into a test plasmid at a location upstream of
LTR-neo sequence.  With this construction, the transfection efficiency
increased 50 to 100 fold, independent of the fragment orientation.
Deletion analysis indicated that the enhancer activity was localized in a
region of less than 1.5kb.  Its sequence is currently being determined.
Rearrangements of proviral genomes were also found in many of the
transductant EC cell clones.

More enhancer-like elements will be isolated from other clones, their
structures determined, activity characterized, and the cellular genes under
the control of these enhancer elements will be identified.  The structure
and biological activity of the rearranged proviral regulatory sequences
will also be determined.  These studies will give us basic insights how
gene expression is controlled in mammalian embryonic stem cells and will
provide us with a means to construct a new series of expression vectors
useful for gene therapy.

## Key facts

- **NIH application ID:** 3178919
- **Project number:** 5R01CA039652-02
- **Recipient organization:** JACKSON LABORATORY
- **SAM.gov UEI:** XR6LMXNKDJJ1
- **Principal Investigator:** MAKOTO TAKETO
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1987
- **Award amount:** $137,424
- **Award type:** 5
- **Project period:** 1986-09-01 → 1989-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3178919

## Citation

> US National Institutes of Health, RePORTER application 3178919, VIRAL GENE EXPRESSION IN EMBRYONAL CARCINOMA CELLS (5R01CA039652-02). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3178919. Licensed CC0.

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