Bacterial and Pneumocystis Infections in the Lungs of Gene-Knockout Rabbits with Severe Combined Immunodeficiency

Song, Jun; Wang, Guoshun; Hoenerhoff, Mark J.; Ruan, Jinxue; Yang, Dongshan; Zhang, Jifeng; Yang, Jie; Lester, Patrick A.; Sigler, Robert E.; Bradley, Michael; Eckley, Samantha S.; Cornelius, Kelsey; Chen, Kong; Kolls, Jay K.; Li, Peng; Ma, Liang; Chen, Yuqing Eugene; Sun, Fei; Xu, Jie

doi:10.3389/fimmu.2018.00429

Cited by 20 publications

(18 citation statements)

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“…Disease-causing mutations were targeted in IL2RG and SPR genes in rabbit cells. Recently, IL2RG-knockout rabbits have been produced as models for human X-linked severe combined immunodeficiency (SCID-X1) 16,17 . The present work shows that patient IL2RG mutations (e.g., C231Y and Q235X) can be efficiently generated in rabbit cells, demonstrating the feasibility of creating SCID-X1 rabbit models carrying patient mutations.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Jang

Chen³

et al. 2019

JoVE

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Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

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Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Jang

Chen³

et al. 2019

JoVE

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“…Four P. oryctolagi DNA samples were obtained from previous studies of one rabbit with severe combined immunodeficiency at the University of Michigan, Ann Arbor, MI, USA (65), and three immunosuppressed rabbits at the Institut Pasteur de Lille (66) Genome assembly was performed essentially as previously described (1,68); detailed analyses of these genomes will be published separately. RNA-Seq analysis of different msg families in P. murina and P. carinii.…”

Section: Methodsmentioning

confidence: 99%

Diversity and Complexity of the Large Surface Protein Family in the Compacted Genomes of MultiplePneumocystisSpecies

Chen

Huang

et al. 2020

mBio

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Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multicopy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins and provide a detailed description of the classification, unique characteristics, and phylogenetic relatedness of five Msg families. We further describe, for the first time, the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests multiple functions for this superfamily rather than just conferring antigenic variation to allow immune evasion as previously believed. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology. IMPORTANCE Pneumocystis continues to be a major cause of disease in humans with immunodeficiency, especially those with HIV/AIDS and organ transplants, and is being seen with increasing frequency worldwide in patients treated with immunode-

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“…Four P. oryctolagi DNA samples were obtained from previous studies of one rabbit with severe combined immunodeficiency at the University of Michigan, Ann Arbor, Michigan, USA (65), and three immunosuppressed rabbits at the Institut Pasteur de Lille (66) and the Institut National de la Recherche Agronomique de Tours Pathologie Aviare et Parasitologie, Tours (67), France.…”

Section: Methodsmentioning

confidence: 99%

Diversity and complexity of the large surface protein family in the compacted genomes of variousPneumocystisspecies

Chen

Huang

et al. 2019

Preprint

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Supplemental Materials: 1 Text file (Text S1) 43 2 Tables (S1-S2) 44 13 Figures (S1-S13) 45 8 Data Sets (Msg sequences for different families) 46 47 48 Pneumocystis, a major opportunistic pathogen in patients with a broad range of 49 immunodeficiencies, contains abundant surface proteins encoded by a multi-copy gene family, 50 termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been 51 identified in all Pneumocystis species characterized to date, highlighting its important role in 52 Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 53 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny 54 and evolution of conserved domains in Msg proteins, and provide detailed description of the 55 classification, unique characteristics and phylogenetic relatedness of five Msg families. We 56 further describe the relative expression levels of individual msg families in two rodent 57 Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from 58 laboratory and wild rats, and the distinct features of the expression site for the classic msg 59 genes in Pneumocystis from 8 mammalian host species. Our analysis suggests a wide variety 60 of functions for this superfamily, not only conferring antigenic variation to allow immune evasion 61 but also mediating life-stage development, optimizing cell mobility and adhesion, and adapting 62 to specific host niches or environmental conditions. This study provides a rich source of 63 information that lays the foundation for the continued experimental exploration of the functions 64 of the Msg superfamily in Pneumocystis biology. 66Pneumocystis continues to be a major cause of disease in humans with 67 immunodeficiencies, especially those with HIV/AIDS and organ transplants, and is being seen 68 with increasing frequency in patients treated with immunodepleting monoclonal antibodies. As 69 an atypical fungus, Pneumocystis has highly adapted to the mammalian lung environment (1), 70 with a high level of host specificity; P. jirovecii infects humans, P. carinii infects Norway rats 71 (Rattus norvegicus), and P. murina infects house mice (Mus musculus). In addition, 72Pneumocystis cell walls are structurally unique and differ significantly from typical fungal cell 73 walls that are comprised of polysaccharides (mainly glucan and chitin) and highly mannosylated 74 proteins. Both genomic and experimental analyses have shown the absence of chitin and outer 75 chain N-mannans in Pneumocystis cell walls (1). Furthermore, beta-1,3-glucan is absent in the 76 trophic form while present but masked in the cyst form of Pneumocystis (2). 77An integral component of the Pneumocystis cell wall in both the cyst and trophic forms is 78 the major surface glycoprotein (Msg) (also known as gp95, gp115, gp120 and gpA) (3-8). Ever 79 since its identification in 1982 (9), Msg has been a focus of research, in part because it is the 80 most abundant Pneumocystis protein as as...

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Bacterial and Pneumocystis Infections in the Lungs of Gene-Knockout Rabbits with Severe Combined Immunodeficiency

Cited by 20 publications

References 30 publications

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Diversity and Complexity of the Large Surface Protein Family in the Compacted Genomes of MultiplePneumocystisSpecies

Diversity and complexity of the large surface protein family in the compacted genomes of variousPneumocystisspecies

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