Expression and Integration of Exogenous DNA Sequences Transfected Into Mammalian Cells

An in vivo approach has been developed for generation of artificial chromosomes, based on the induction of intrinsic, large-scale amplification mechanisms of mammalian cells. Here, we describe the successful generation of prototype human satellite DNA-based artificial chromosomes via amplification-dependent de novo chromosome formations induced by integration of exogenous DNA sequences into the centromeric/rDNA regions of human acrocentric chromosomes. Subclones with mitotically stable de novo chromosomes were established, which allowed the initial characterization and purification of these artificial chromosomes. Because of the low complexity of their DNA content, they may serve as a useful tool to study the structure and function of higher eukaryotic chromosomes. Human satellite DNA-based artificial chromosomes containing amplified satellite DNA, rDNA, and exogenous DNA sequences were heterochromatic, however, they provided a suitable chromosomal environment for the expression of the integrated exogenous genetic material. We demonstrate that induced de novo chromosome formation is a reproducible and effective methodology in generating artificial chromosomes from predictable sequences of different mammalian species. Satellite DNA-based artificial chromosomes formed by induced large-scale amplifications on the short arm of human acrocentric chromosomes may become safe or low risk vectors in gene therapy.

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Patterns of integration of exogenous DNA sequences transfected into mammalian cells of primate and rodent origin

Gene ◽

10.1016/0378-1119(86)90332-x ◽

1986 ◽

Vol 50 (1-3) ◽

pp. 279-288 ◽

Cited By ~ 19

Author(s):

Florence Colbère-Garapin ◽

Marie-Louise Ryhiner ◽

Isabelle Stephany ◽

Philippe Kourilsky ◽

Axel-Claude Garapin

Keyword(s):

Dna Sequences ◽

Mammalian Cells ◽

Exogenous Dna

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Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4185 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4185-4189 ◽

Cited By ~ 7

Author(s):

J A Greenspan ◽

F M Xu ◽

R L Davidson

Keyword(s):

Cell Line ◽

Cell Lines ◽

Dna Sequences ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Shuttle Vector ◽

Ethyl Methanesulfonate ◽

Wild Type ◽

Single Base ◽

Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

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A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

Nucleic Acids Research ◽

10.1093/nar/gkw707 ◽

2016 ◽

pp. gkw707 ◽

Cited By ~ 9

Author(s):

Brian Chaikind ◽

Jeffrey L. Bessen ◽

David B. Thompson ◽

Johnny H. Hu ◽

David R. Liu

Keyword(s):

Fusion Protein ◽

Dna Sequences ◽

Mammalian Cells ◽

Serine Recombinase

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Distribution, expression and germ line transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs

Development ◽

10.1242/dev.99.1.15 ◽

1987 ◽

Vol 99 (1) ◽

pp. 15-23

Author(s):

L.D. Etkin ◽

B. Pearman

Keyword(s):

Xenopus Laevis ◽

Dna Sequences ◽

Copy Number ◽

Germ Line ◽

Gene Promoter ◽

Early Gene ◽

Mosaic Pattern ◽

Exogenous Dna ◽

Gene Coding ◽

Germ Line Transmission

We analysed the fate, expression and germ line transmission of exogenous DNA which was microinjected into fertilized eggs of Xenopus laevis. DNA was injected into fertilized eggs within 1 h following fertilization. The injected DNA was dispersed around the site of injection and became localized to cleavage nuclei by stage 6. Injected DNA persisted in the tissues of 6- to 8-month-old frogs and exhibited a mosaic pattern of distribution with regard to the presence or absence and copy number between different tissues. We detected the exogenous DNA sequences in 60% of injected frogs. Restriction digestion analysis of this DNA suggested that it is not rearranged and was organized as head-to-tail multimers. The copy number varied from 2 to 30 copies/cell in various tissues of the same frog. Plasmid pSV2CAT which contains the prokaryotic gene coding for chloramphenicol acetyl transferase (CAT) enzyme linked to the SV40 early gene promoter was expressed in 50% of the animals containing the gene. The pattern of expression, however, varied between different animals and could not be correlated with copy number. We also showed that the exogenous DNA sequences were transmitted through the male germ line and that each offspring contained the gene integrated into a different region of the genome.

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Gene replacement with one-sided homologous recombination

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.360-367.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 360-367

Author(s):

N Berinstein ◽

N Pennell ◽

C A Ottaway ◽

M J Shulman

Keyword(s):

Homologous Recombination ◽

Dna Sequences ◽

Mammalian Cells ◽

Target Gene ◽

Gene Replacement ◽

Immunoglobulin Genes ◽

Nonhomologous Recombination ◽

Immunoglobulin Locus ◽

Chromosomal Sequences ◽

Dna Vectors

Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.

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The search for homology does not limit the rate of extrachromosomal homologous recombination in mammalian cells.

Genetics ◽

10.1093/genetics/136.2.597 ◽

1994 ◽

Vol 136 (2) ◽

pp. 597-605

Author(s):

A S Waldman

Keyword(s):

Homologous Recombination ◽

Dna Sequences ◽

Mammalian Cells ◽

Insertion Mutant ◽

Insertion Mutation ◽

Homologous Pairing ◽

Rate Limiting Step ◽

Limiting Step ◽

Homology Searching ◽

Rate Limiting

Abstract Mouse LTK- cells were transfected with a pair of defective Herpes simplex virus thymidine kinase (tk) genes. One tk gene had an 8-bp insertion mutation while the second gene had a 100-bp inversion. Extrachromosomal homologous recombination leading to the reconstruction of a functional tk gene was monitored by selecting for tk positive cells using medium supplemented with hypoxanthine/aminopterin/thymidine. To assess whether the search for homology may be a rate-limiting step of recombination, we asked whether the presence of an excess number of copies of a tk gene possessing both the insertion and inversion mutations could inhibit recombination between the singly mutated tk genes. Effective competitive inhibition would require that homology searching (homologous pairing) occur rapidly and efficiently. We cotransfected plasmid constructs containing the singly mutated genes in the presence or absence of competitor sequences in various combinations of linear or circular forms. We observed effective inhibition by the competitor DNA in six of the seven combinations studied. A lack of inhibition was observed only when the insertion mutant gene was cleaved within the insertion mutation and cotransfected with the two other molecules in circular form. Additional experiments suggested that homologous interactions between two DNA sequences may compete in trans with recombination between two other sequences. We conclude that homology searching is not a rate-limiting step of extrachromosomal recombination in mammalian cells. Additionally, we speculate that a limiting factor is involved in a recombination step following homologous pairing and has a high affinity for DNA termini.

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Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA

Development ◽

10.1242/dev.102.2.287 ◽

1988 ◽

Vol 102 (2) ◽

pp. 287-299 ◽

Cited By ~ 2

Author(s):

R.R. Franks ◽

B.R. Hough-Evans ◽

R.J. Britten ◽

E.H. Davidson

Keyword(s):

Dna Sequences ◽

Larval Growth ◽

Exogenous Dna ◽

Zygote Nucleus ◽

Cell Lineages ◽

Larval Stages ◽

Injection Process ◽

End To End ◽

Nuclear Injection ◽

Cytoplasmic Injection

A method is described for microinjection of cloned DNA into the zygote nucleus of Lytechinus variegatus. Eggs of this species are unusually transparent, facilitating visual monitoring of the injection process. The initial fate of injected DNA fragments appears similar to that observed earlier for exogenous DNA injected into unfertilized egg cytoplasm. Thus after end-to-end ligation, it is replicated after a lag of several hours to an extent indicating that it probably participates in most of the later rounds of DNA synthesis undergone by the host cell genomes during cleavage. The different consequences of nuclear versus cytoplasmic injection are evident at advanced larval stages. Larvae descendant from eggs in which exogenous DNA was injected into the nuclei are four times more likely (32% versus 8%) to retain this DNA in cell lineages that replicate very extensively during larval growth, i.e. the lineages contributing to the imaginal rudiment, and thus to display greatly enhanced contents of the exogenous DNA. Similarly, 36% of postmetamorphic juveniles from a nuclear injection sample retained the exogenous DNA sequences, compared to 12% of juveniles from a cytoplasmic injection sample. However, the number of copies of the exogenous DNA sequences retained per average genome in postmetamorphic juveniles was usually less than 0.1 (range 0.05-50), and genome blot hybridizations indicate that these sequences are organized as integrated, randomly oriented, end-to-end molecular concatenates. It follows that only a small fraction of the cells of the average juvenile usually retains the exogenous sequences. Thus, even when introduced by nuclear microinjection, the stable incorporation of exogenous DNA in the embryo occurs in a mosaic fashion, although in many recipients the DNA enters a wider range of cell lineages than is typical after cytoplasmic injection. Nuclear injection would probably be the route of choice for studies of exogenous DNA function in the postembryonic larval rudiment.

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