Transfer of genes into human somatic cells using retrovirus vectors.
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Animals↗
Biomedical subjects
A D Miller
Publications and source records attributed to A D Miller.
Recombinant retroviruses in transgenic mice.
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Animals↗
Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.
Retrovirus vectors can be made in the absence of helper virus by using retrovirus packaging cell lines. Helper-free virus is critical for a variety of gene transfer studies. The most useful packaging cell lines contain helper virus DNA from which the signal required for packaging of the viral RNA genome into virions has been deleted. However, we showed that the ability to package virus is conferred at very low frequency to cells infected with virus from these packaging cell lines, presumably by low-frequency transmission of the deleted virus genome. In addition, these packaging cell lines can interact with some retroviral vectors to yield replication-competent virus. We constructed packaging cell lines containing helper virus DNA that had several alterations in addition to deletion of the packaging signal. The new packaging cells retained the useful features of previously available lines but did not yield helper virus after introduction of any of the vectors tested, and transfer of the packaging function was not detected.
Cell Line↗
Removal of a 67-base-pair sequence in the noncoding region of protooncogene fos converts it to a transforming gene.
Transformation of fibroblasts by protooncogene fos (c-fos) requires the linkage of viral long terminal repeat (LTR) sequences and interruption of 3'-noncoding sequences. We have identified an A + T-rich stretch of 67 nucleotides, located 627-693 base pairs downstream from the coding domain and 123-189 base pairs upstream from the putative poly(A) addition site, removal of which confers transforming activity to the c-fos gene. A novel regulation of the expression of the c-fos gene is proposed, which may be functional in vivo to prevent the gene from becoming an oncogene.
Animals↗
Efficient insertion of genes into the mouse germ line via retroviral vectors.
We present a general strategy for the efficient insertion of recombinant retroviral vector DNA into the mouse germ line via infection of preimplantation mouse embryos. Transgenic mice were generated that harbor a replication-competent recombinant retrovirus (delta Mo + Py M-MuLV) that lacks the Moloney murine leukemia virus (M-MuLV)-type enhancer sequence in the long terminal repeat (LTR). Instead, the LTR contains an enhancer element that permits polyoma virus F101 to grow in undifferentiated F9 embryonal carcinoma cells. Expression studies in different tissues of animals transgenic for delta Mo + Py M-MuLV indicate possibilities to target and modulate expression of retroviral recombinants in mice via their LTR enhancer sequences. In addition, 16 transgenic mice were generated that harbor proviral DNA of a defective recombinant retrovirus carrying a mutant dihydrofolate reductase gene.
Animals↗
Deletion of the gag region from FBR murine osteosarcoma virus does not affect its enhanced transforming activity.
Both FBJ murine osteosarcoma virus (FBJ-MSV) and FBR-MSV induce transformation in tissue culture and osteogenic sarcomas in mice. In tissue culture, however, FBR-MSV induces larger foci with a shorter latency than those induced by FBJ-MSV. Transformation is dependent on expression of the fos oncogene in FBJ-MSV and of a gag-fos fusion protein in FBR-MSV. We have determined that the gag sequences can be deleted from FBR-MSV without affecting the high transforming activity of this virus in comparison to FBJ-MSV. The resultant virus, designated FBJ/R-MSV, has a protein coding region that is half the size of that of FBR-MSV and about one-third smaller than that of FBJ-MSV. Thus, FBJ/R-MSV will provide a useful tool for studying the transforming activity of the fos oncogene.
Animals↗
Generation of helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase gene.
We constructed several retroviruses which transduced a mutant dihydrofolate reductase gene that was resistant to methotrexate inhibition and functioned as a dominant selectable marker. The titer of dihydrofolate reductase-transducing virus produced by virus-producing cells could be increased to very high levels by selection of the cells in increasing concentrations of methotrexate. Helper virus-free dihydrofolate reductase-transducing virus was also generated by using a broad-host-range amphotropic retroviral packaging system. Cell lines producing helper-free dihydrofolate reductase-transducing virus with a titer of 4 X 10(6) per ml were generated. These retroviral vectors should have general utility for high-efficiency transduction of genes in cultured cells and in animals.
Animals↗
Responses to head tilt in cat central vestibular neurons. II. Frequency dependence of neural response vectors.
The responses of central vestibular neurons in the decerebrate cat subjected to whole-body tilt were examined as a function both of stimulus orientation (with respect to the cat's head) and frequency, with the aim of understanding the neural processing responsible for producing the observed response patterns. Responses to whole-body tilt were recorded from vestibular neurons in and around the lateral vestibular nucleus (LVN). By plugging all six semicircular canals, the otolith contribution was studied in isolation. For each neuron, a response vector was defined as having three components: orientation, gain, and phase. These components were examined using sinusoidal stimulus frequencies of 0.01 to 2 Hz. The orientation component of the neural response vector does not vary as a function of stimulus frequency. Thus response dynamics previously described with a fixed (roll) axis cannot be explained by changes in the angle between the response vector orientation and a fixed stimulus axis. Two major classes of neural responses were observed. One class had a phase lead at low frequencies and gain that showed a modest increase with frequency. It could be described by a model that included a fractional s exponent operator. These response dynamics resemble that of otolith afferents, suggesting that these neurons may be acting as simple relays. The other major response class was characterized by a large gain increase and a phase lag of as much as 180 degrees as frequency increased; such response dynamics have been previously observed in otolith-evoked neck and forelimb reflexes. A more complex model, consisting of a parallel excitatory and high-pass-filtered inhibitory limb, was necessary to describe these responses. The orientation component of the response vector of most of the neurons whose dynamics were best described by the parallel pathway model pointed toward the contralateral side, implying they would be excited by side-up tilt (at low frequencies). Most other neurons had ipsilateral vectors.
Animals↗
Control of abdominal muscles by brain stem respiratory neurons in the cat.
Control of abdominal musculature by brain stem respiratory neurons was studied in decerebrate unanesthetized cats by determining 1) which brain stem respiratory neurons could be antidromically activated from the lumbar cord, from which the abdominal muscles receive part of their innervation, and 2) if lumbar-projecting respiratory neurons make monosynaptic connections with abdominal motoneurons. A total of 462 respiratory neurons, located between caudal C2 and the retrofacial nucleus (Bötzinger complex), were tested for antidromic activation from the upper lumbar cord. Fifty-eight percent of expiratory (E) neurons (70/121) in the caudal ventral respiratory group (VRG) between the obex and rostral C1 were antidromically activated from contralateral L1. Eight of these neurons were activated at low thresholds from lamina VIII and IX in the L1-2 gray matter. One-third (14/41) of the E neurons that projected to L1 could also be activated from L4-5. Almost all antidromic E neurons had an augmenting firing pattern. Ten scattered inspiratory (I) neurons projected to L1 but could not be activated from L4-5. No neurons that fired during both E and I phases (phase-spanning neurons) were antidromically activated from the lumbar cord. In order to test for possible monosynaptic connections between descending E neurons and abdominal motoneurons, cross-correlations were obtained between 27 VRG E neurons, which were antidromically activated from caudal L2 and contralateral L1 and L2 abdominal nerve activity (47 neuron-nerve combinations). Only two neurons showed a correlation with one of the two nerves tested. Although there is a large projection to the lumbar cord from expiratory neurons in the ventral respiratory group caudal to the obex, cross-correlation analyses suggest that strong monosynaptic connections between these neurons and abdominal motoneurons are scarce.
Abdominal Muscles↗
Diabetes-related lower extremity amputations in New Jersey, 1979 to 1981.
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Adult↗
Infectious and selectable retrovirus containing an inducible rat growth hormone minigene.
A growth hormone minigene carrying its natural promoter (237 nucleotides of chromosomal DNA) was stably propagated in a murine retrovirus containing hypoxanthine-guanine phosphoribosyltransferase as a selectable marker. Glucocorticoid and thyroid hormone inducibility was transferred with the growth hormone gene. Recombinant virus with titers of 10(6) per milliliter was recovered. This demonstration that retroviruses can be used to transfer a nonselectable gene under its own regulatory control enlarges the scope of retroviral vectors as potent tools for gene transfer.
Animals↗
Expression of a retrovirus encoding human HPRT in mice.
Transmissible retroviruses encoding human hypoxanthine phosphoribosyltransferase (HPRT) were used to infect mouse bone marrow cells in vitro, and the infected cells were transplanted into mice. Both active human HPRT-protein and chronic HPRT-virus production were detected in hematopoietic tissue of the mice, showing transfer of the gene. These results indicate the possible use of retroviruses for somatic cell therapy.
Animals↗
Partial phenotypic correction of human Lesch-Nyhan (hypoxanthine-guanine phosphoribosyltransferase-deficient) lymphoblasts with a transmissible retroviral vector.
A human Lesch-Nyhan (hereditary, severe hypoxanthine-guanine phosphoribosyltransferase (HPR transferase) deficiency) B-lymphoblast line was infected with an amphotropic retroviral vector containing human HPR transferase cDNA under transcriptional control of viral long terminal repeat sequences. Of 17 clones isolated, 12 integration groups were defined by analysis of restriction enzyme digests of their genomic DNA with HPR transferase and viral long terminal repeat probes. These groups had HPR transferase activity restored to levels of 4 to 23% of normal values. Aberrant metabolic parameters associated with severe deficiency of HPR transferase activity, i.e. elevated rates of purine excretion, increased accumulation of hypoxanthine, elevated 5-phosphoribosyl-1-pyrophosphate contents, altered nucleoside triphosphate pools, resistance to toxic effects of 6-thioguanine, were partially to nearly completely corrected; the degree of correction generally corresponded to the degree of restoration of HPR transferase activity. The integration of the HPR transferase gene was found to be variably stable during 9 months of culture of the virally transformed lymphoblasts under nonselective conditions. The HPR transferase gene-infected lines reverted to resistance to 20 microM 6-thioguanine, i.e. severe HPR transferase deficiency, at frequencies of 10(-6) to in excess of 10(-5) per generation. The reversions were accompanied by either a loss or rearrangement of the integrated HPR transferase sequences or by retention of the sequences in an unaltered form.
Animals↗
Ethics and administration.
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Ethics, Professional↗
c-fos protein can induce cellular transformation: a novel mechanism of activation of a cellular oncogene.
The FBJ murine osteosarcoma virus (FBJ-MuSV) induces tumors in vivo and transformation in vitro. Transformation is due to the expression of a single viral protein (p55v-fos) which is encoded by sequences derived from mouse genetic material. The homologous cellular gene (c-fos) does not transform cultured cells after introduction by transfection. We show that even though the c-fos protein is completely different from the v-fos protein at its C terminus, it is capable of transforming cultured fibroblasts. However, activation of the transforming potential of the c-fos gene requires two manipulations--a transcriptional enhancer sequence must be linked to the gene and an interaction at the 3' end of the gene, which inhibits transformation, must be disrupted. Our studies show that normal cellular protein can induce transformation when expressed in an inappropriate cell type.
Animals↗
Viral and cellular fos proteins: a comparative analysis.
The FBJ murine osteosarcoma virus (FBJ-MuSV) induces osteosarcomas in mice and transforms fibroblasts in vitro. It contains an oncogene termed v-fos derived from a normal cellular gene by recombination with an associated helper virus. The product of the v-fos gene is a 55,000 dalton protein, p55v-fos. This protein was found in the nuclei of cells containing amplified levels of the v-fos gene, and also in the nuclei of virus-transformed cells. The c-fos protein was localized in the nuclei of normal mouse amnion cells and in the nuclei of cells transformed by a recombinant plasmid that expresses the c-fos gene product. However, p55c-fos undergoes more extensive post-translational modification in the nucleus than p55v-fos. Immunofluorescence data indicate that the level of p55c-fos in normal mouse amnion cells is similar to that found in fibroblasts transformed by the v-fos or c-fos proteins.
Amnion↗
Two base changes restore infectivity to a noninfectious molecular clone of Moloney murine leukemia virus (pMLV-1).
The complete nucleotide sequence of a molecular clone of Moloney murine leukemia virus (pMLV-1) has previously been reported (Shinnick et al., Nature [London] 293:543-548, 1981). However, pMLV-1 does not generate infectious virus after transfection into cells (Berns et al., J. Virol. 36:254-263, 1980). The lesion in pMLV-1 has been localized by determining the biological activity of recombinants containing DNA from an infectious clone of Moloney murine leukemia virus (pMLV-48) and pMLV-1. Replacement of a 1.0-kilobase pair region which spans the gag-pol junction of pMLV-1 with the corresponding DNA fragment from the infectious clone restores its infectivity. Nucleotide sequence analysis of this fragment obtained from the infectious clone (pMLV-48) and pMLV-1 reveals two single base pair changes, one in the p30gag gene and the other in the 5' end of the pol gene. The mutation in the pol gene does not affect the production of infectious virus but renders them XC negative, whereas the mutation in the gag gene appears to be lethal. The complete nucleotide sequence of an infectious clone of Moloney murine leukemia virus can now be deduced.
Base Sequence↗
Responses to head tilt in cat central vestibular neurons. I. Direction of maximum sensitivity.
Responses to head tilt were recorded from vestibular neurons in and around the lateral vestibular nucleus (LVN) of the decerebrate cat. Each animal had all six semicircular canals rendered nonfunctional by a plugging procedure. Each cell was studied by slowly tilting the cat, using one or both of two paradigms. In the first method, sinusoidal tilts (0.05 or 0.1 Hz) were used to produce bidirectional stimuli in up to 12 pairs of directions, including left/right (roll tilt) and fore/aft (pitch). The second method imposed a constant 10 degree tilt; the direction of the tilt was rotated around the animal by an appropriate combination of roll and pitch motions. Neurons responded by maximally increasing their discharge frequency in a particular direction of head tilt from the horizontal. Each cell's response could be described by a vector in the animal's horizontal plane whose orientation is given by the direction of the most effective stimulus and whose length represents the neuron's maximal sensitivity to tilt. The two methods of stimulation yielded equivalent response vectors. Response vectors were obtained for 100 neurons. The distribution of vector directions for these vestibular neurons was not uniform; there was a conspicuous absence of neurons with fore/aft-directed vectors. The sensitivity of these cells (length of the response vector) ranged from 10 to 230 impulses X s-1 X g-1 (median 50). Neurons whose vectors lay in the ipsilateral half-plane (which would be excited by ear-down tilt) tended to be less sensitive than those with contralateral vectors. Neurons excited by ear-up tilt tended to be located ventrally in the LVN, while those excited by ear-down tilt were more evenly distributed. There was no other obvious correlation of vector orientation with the anatomical locus of the cell in the LVN. The directional selectivity of the responses of these neurons to head tilts are similar to those previously reported tin utricular afferents. The broad distribution of response vector orientations provides an appropriate substrate for vestibulospinal reflexes to a wide variety of head tilts.
Afferent Pathways↗