Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi

Armaleo, Daniele; Ye, Guang Ning; Klein, Theodore M.; Shark, Katherine B.; Sanford, Jay P.; Johnston, Stephen Albert

doi:10.1007/bf00312852

Cited by 156 publications

(64 citation statements)

References 15 publications

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“…With the biolistic system, both circular and linear ade2 generated similar numbers of transformants per plate: 20 to 30 colonies with ade2 cDNA and 103 to 104 colonies with genomic ade2. This finding concurs with biolistic transformation of S. cerevisiae, in which both circular and linear DNAs had similar transformation frequencies (2). However, these results are quite different from those with electroporation, in which only the linear ade2 generated a significant number of aade2-27 transformants (Table 1).…”

Section: Discussionsupporting

confidence: 83%

“…C. neofonnans cells were prepared for electroporation as described previously by Varma et al (31) and were prepared for biolistics by using an adaptation of a method described for S. cerevisiae (2). Briefly, cells from an early-stationary-phase culture in YEPD were diluted 1:50 in fresh YEPD and grown with vigorous shaking at 30°C for either 5 to 6 h (for electroporation) or 18 to 24 h (for biolistics).…”

Section: Strainsmentioning

confidence: 99%

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Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA

et al. 1993

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A transformation scheme for Cryptococcus neoformans to yield high-frequency, integrative events was developed. Adenine auxotrophs from a clinical isolate of C. neoformans serotype A were complemented by the cryptococcal phosphoribosylaminoimidazole carboxylase gene (ade2) with a biolistic DNA delivery system.Comparison of two DNA delivery systems (electroporation versus a biolistic system) showed notable differences. The biolistic system did not require linear vectors and transformed each auxotrophic strain at similar fr-equencies. Examination of randomly selected transformants by biolistics showed that 15 to 40%O were stable, depending on the recipient auxotroph, with integrative events identified in all stable transformants by DNA analysis. Although the ade2 cDNA copy transformed at a low frequency, DNA analysis found homologous recombination in each of these transformants. DNA analysis of stable transformants receiving genomic ade2 revealed ectopic integration in a majority of cases, but approximately a quarter of the transformants showed homologous recombination with vector integration or gene replacement. This system has the potential for targeted gene disruption, and its efficiency will also allow for screening of DNA libraries within C. neoformans. Further molecular strategies to study the pathobiology of this pathogenic yeast are now possible with this transformation system.Cryptococcus neoformans is a pathogenic yeast with an unexplained predilection for infecting the central nervous system. There has been a significant increase in the number of human infections with this yeast over the last decade as the immunocompromised population has enlarged because of AIDS. Although epidemiological studies suggest that the increase in cryptococcal infection is due to more-susceptible hosts rather than increased virulence, the pathobiology of the organism needs further investigation. Several phenotypic factors associated with virulence have been identified for C. neoformans: the polysaccharide capsule (10,16,20), melanin production (19,20,26), and growth at 370C (20,26 Recent studies of C neofonnans have established basic molecular techniques for performing site-directed mutagenesis (9, 31). A C. neoformans uraS auxotrophic strain (serotype D) was recently reported to be transformed at a low efficiency by complementation with a cloned uraS gene delivered by electroporation. However, the number of integrative events was low and generally nonhomologous (9, 31). A total of 80 to 90% of the transformants were unstable and apparently contained extrachromosomal DNA, consisting of sequences of rearranged uraS-vector DNA with the addition of cryptococcal genomic DNA (8,9,31

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Section: Discussionsupporting

confidence: 83%

Section: Strainsmentioning

confidence: 99%

Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA

et al. 1993

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“…The two plasmids were either coprecipitated on the same gold particles (ϭTOGETHER) to ensure that both epitopes will be expressed by the same cell (11) or each plasmid was precipitated on separate gold particles (ϭSEPARATE) to ensure that only one epitope would be expressed by each host cell (11). Mice immunized with both plasmids produced robust CTL activity (Fig.…”

Section: Coimmunization With Mutual In Vitro Antagonists Does Not Resmentioning

confidence: 99%

The Role of T Cell Antagonism and Original Antigenic Sin in Genetic Immunization

Singh

Rodgers

Barry

2002

The Journal of Immunology

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To counter highly mutable pathogens like HIV-1, a number of vaccines are being developed to deliver multiple mutant forms of viral Ags to provoke multivalent antiviral CTLs. However, it is uncertain whether such multiple mutant epitope vaccines will generate the diverse CTL responses desired or will instead create immune interference. To characterize the role of immune interference by mutant epitopes in this process, we have tested a “worst case” scenario in which the immunodominant epitope of OVA (SIINFEKL) and its in vitro TCR antagonist (SIINFEDL) have been used to genetically immunize C57BL/6 mice. We demonstrate here that sequential delivery of these mutant epitopes provokes original antigenic sin in CD8 T cells as demonstrated by attenuation of CTLs, intracellular IFN-γ production, and MHC I peptide-tetramer staining. By contrast, simultaneous exposure of the immune system to this agonist/antagonist pair not only fails to generate T cell antagonism in vivo, but also avoids original antigenic sin. These observations suggest that simultaneous immunization with vaccines containing mutant epitopes, even T cell antagonists, can indeed generate a diverse array of T cell responses and that at least some immune interference can be avoided by delivering mutant Ags to the immune system simultaneously.

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“…These are very restricted, so far (Davis et al, 1993;Zhang et al, 1999). The genetic shotgun was introduced for gene delivery in 1990 (Armaleo et al, 1990). In this protocol, microscopic tungsten spheres are used to deliver DNA or RNA constructs.…”

Section: Delivery Of Nucleotides In To Es Cellsmentioning

confidence: 99%

Embryonic Stem Cells: Introducing Exogenous Regulators into Embryonic Stem Cells

Cheon¹

2011

Embryonic Stem Cells - Basic Biology to Bioengineering

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Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi

Cited by 156 publications

References 15 publications

Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA

Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA

The Role of T Cell Antagonism and Original Antigenic Sin in Genetic Immunization

Embryonic Stem Cells: Introducing Exogenous Regulators into Embryonic Stem Cells

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