Publications by authors named "Xien Chen"

8 Publications

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Expanding the Toolkit for Genome Editing in a Disease Vector, Transgenic Lines Expressing Cas9 and Single Guide RNA Induce Efficient Mutagenesis.

CRISPR J 2021 Jan 15. Epub 2021 Jan 15.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA.

CRISPR-Cas9 mediated genome editing methods are being used for the analysis of gene function. However, it is hard to identify gene knockout mutants for genes whose knockout does not cause distinct phenotypes. To overcome this issue in the disease vector, , a transgenic Cas9/single guide RNA (sgRNA) method, was used to knock out the eye marker gene, (), and the juvenile hormone receptor, (). PiggyBac transformation vectors were prepared to express sgRNAs targeting and under the control of the U6 promoter. Transgenic expressing -sgRNA or -sgRNA under the control of the U6 promoter and enhanced green fluorescent protein (eGFP) under the control of the hr5ie1 promoter were produced. The U6-sgRNA adults were mated with AAEL010097-Cas9 adults. The progeny were screened, and the insects expressing eGFP and DsRed were selected and evaluated for mutations in target genes. About 77% and 78% of the progeny that were positive for both eGFP and DsRed in -sgRNA and -sgRNA groups, respectively, showed mutations in their target genes.
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http://dx.doi.org/10.1089/crispr.2020.0052DOI Listing
January 2021

systemic RNA interference defective protein 1 enhances RNAi efficiency in a lepidopteran insect, the fall armyworm, in a tissue-specific manner.

RNA Biol 2020 Nov 9:1-9. Epub 2020 Nov 9.

Department of Botany, Kongunadu Arts and Science College (Autonomous), Bharathiar University, Coimbatore, India.

RNA interference (RNAi) is an important tool for gene function studies in insects, especially in non-model insects. This technology is also being developed for pest control. However, variable RNAi efficiency among insects is limiting its use in insects. Systemic RNAi in requires systemic RNA interference defective protein 1 (. The expression of Ce in insect cell lines was shown to improve RNAi. However, the mechanisms through which this double-stranded RNA (dsRNA) transporter improves RNAi efficiency in insects is not known. We stably expressed in two cell lines, Sf9 and Sf17 cells derived from ovary and midgut, respectively. Expression of enhanced RNAi efficiency in ovarian Sf9 cells, but not in midgut Sf17 cells. Reduced accumulation of dsRNA in late endosomes and successful processing dsRNA to siRNA contribute to enhanced RNAi efficiency in Sf9 cells. Transgenic expressing were produced and tested for RNAi efficiency. RNAi efficiency enhancement due to expression showed tissue specificity. Compared to RNAi efficiency in wild-type expressing transgenic showed a significant improvement of RNAi in tissues such as Verson's glands. In contrast, no improvement in RNAi was observed in tissues such as midgut. The cell-type specific and tissue-specific enhancement of RNAi efficiency by in provides valuable information for improving RNAi in insects such as those belonging to order Lepidoptera where RNAi is variable and inefficient.
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http://dx.doi.org/10.1080/15476286.2020.1842632DOI Listing
November 2020

Identification and characterization of highly active promoters from the fall armyworm, Spodoptera frugiperda.

Insect Biochem Mol Biol 2020 11 19;126:103455. Epub 2020 Aug 19.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546, United States. Electronic address:

The cell lines derived from the fall armyworm (FAW), Spodoptera frugiperda, have been widely used for production of recombinant proteins for applications in both basic research and applications in medicine and agriculture. Promoters from the nucleopolyhedrovirus (NPV) are commonly used in these expression systems. These promoters have some limitations, which may be overcome by using promoters of genes from S. frugiperda. However, information on these promoters is not available. We identified several highly expressed genes from the transcriptomes of S. frugiperda midgut, fat body, epidermis, ovarian cell line (Sf9), and a midgut cell line (Sf17). The activity of potential promoters of 21 highly expressed genes was evaluated in Sf9 and Sf17 cells. Two of these promoters, SfHSC70-P1780 and SfPub-P2009, showed higher activity than commonly used hr5/ie1 (baculovirus enhancer element, hr5 and immediate early gene 1, ie1) promoter. Interestingly, the activity of these two promoters increased after adding hr5 enhancer element. The hr5/SfPub-P2009 promoter performance was evaluated by expressing an exogenous P450 protein in Sf9 cells using a plasmid-based expression system. The activity of this promoter was also evaluated in the FAW by expressing green fluorescence protein using the baculovirus expression system. In both cases, the hr5/SfPub-P2009 promoter performed better than the commonly used hr5/ie1 promoter. These strong endogenous promoters will be useful for studies in S. frugiperda and other lepidopteran insects for multiple applications, including protein expression, genome editing, and transgenic insects.
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http://dx.doi.org/10.1016/j.ibmb.2020.103455DOI Listing
November 2020

Identification and functional analysis of promoters of heat-shock genes from the fall armyworm, Spodoptera frugiperda.

Sci Rep 2020 02 11;10(1):2363. Epub 2020 Feb 11.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546, United States of America.

The functional information on heat-shock proteins (Hsp) and heat-shock promoters from an important agricultural insect pest, Spodoptera frugiperda, is still lacking. We conducted a genome-wide identification of Hsp genes and identified a total of 21 genes belonging to four major insect Hsp families (small heat-shock proteins, Hsp60, Hsp70, and Hsp90) in S. frugiperda. Expression of most of S. frugiperda (SfHsp) genes could be detected in Sf9 cells, embryos and larval tissues of S. frugiperda. The heat-inducible activity of heat-shock promoters from several SfHsp genes was tested in Sf9 cells and embryos. The promoter of SfHsp70D showed the high constitutive activity in cell line and embryos, while the activity of SfHsp20.15 and SfHsp20.71 promoters was most dramatically induced in Sf9 cells and embryos. In embryos, the heat-induced activity of SfHsp20.71 and SfHsp70D promoters outperformed commercially used ie1 and ie2 promoters. The heat-induced activity of SfHsp70D and SfHsp19.07 promoters were more robust than ie2 promoter in Sf9 cells. These SfHsp promoters with high basal activity or with heat-induced activity from low basal activity, could be used in S. frugiperda or other lepidopteran insects for many applications including transgenesis and genome editing.
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http://dx.doi.org/10.1038/s41598-020-59197-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012861PMC
February 2020

Mutation of doublesex in Hyphantria cunea results in sex-specific sterility.

Pest Manag Sci 2020 May 25;76(5):1673-1682. Epub 2019 Dec 25.

College of Forestry, Northwest A&F University, Yangling, China.

Background: The gene doublesex (dsx) plays pivotal roles in sex determination and controls sexually dimorphic development in certain insects. Importantly, it also displays a potential candidate target for pest management due to its sex-specific splicing. Therefore, we used CRISPR/Cas9-mediated gene disruption to investigate the function of dsx in Hyphantria cunea, an invasive forest pest.

Result: In the present study, we identified the dsx gene from H. cunea which showed a sex-biased expression pattern that was different from other lepidopteran insects. Referring to sex-specific functional analyses in Bombyx mori, we performed a site-specific knockout of the Hcdsx gene by using a CRISPR/Cas9 system, which induced severe abnormalities in external genitalia and some incomplete sex reversal phenotypes, which in turn led to reduced sex-specific fecundity. An alternative splicing pattern of Hcdsx was altered by CRISPR/Cas9-induced mutation, and alterations in splicing affected expression of downstream genes encoding pheromone binding protein 1, vg1 and vg2 (encoding vitellogenin), which contributed to the sex-specific sterility phenotypes in the Hcdsx mutants.

Conclusion: The Hcdsx gene plays important roles in sexual differentiation in H. cunea. Disruption of Hcdsx induced sex-specific sterility, demonstrating a potential application in control of this pest. © 2019 Society of Chemical Industry.
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http://dx.doi.org/10.1002/ps.5687DOI Listing
May 2020

Disruption of sex-specific doublesex exons results in male- and female-specific defects in the black cutworm, Agrotis ipsilon.

Pest Manag Sci 2019 Jun 7;75(6):1697-1706. Epub 2019 Jan 7.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

Background: Doublesex (dsx), the downstream gene in the insect sex-determination pathway, is a key regulator of sexually dimorphic development and behavior across a variety of insects. Manipulating expression of dsx could be useful in the genetic control of insects. However, information on the sex-specific function of dsx in non-model insects is lacking.

Results: In this work, we isolated a dsx homolog, which is alternatively spliced into six female-specific and one male-specific isoforms, from an important agricultural pest, the black cutworm, Agrotis ipsilon. Studies on the expression of sex-specific Aidsx mRNA during embryonic development showed that the sixth hour post oviposition is the key stage for sex determination in A. ipsilon. Functional analysis of Aidsx was conducted using a CRISPR/Cas9 system targeting female- and male-specific Aidsx exons. Disruptions of sex-specific Aidsx exons resulted in sex-specific, sexually dimorphic defects in external genitals, gonads and antennae, and expression of sex-specific genes as well as production of offspring in both sexes.

Conclusion: Our results not only demonstrate that dsx is a key player determining A. ipsilon sexually dimorphic traits, but also provide a potential method for the genetic control of this pest. © 2018 Society of Chemical Industry.
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http://dx.doi.org/10.1002/ps.5290DOI Listing
June 2019

Double-stranded RNA binding protein, Staufen, is required for the initiation of RNAi in coleopteran insects.

Proc Natl Acad Sci U S A 2018 08 30;115(33):8334-8339. Epub 2018 Jul 30.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY 40546

RNA interference (RNAi) is being used to develop methods to control pests and disease vectors. RNAi is robust and systemic in coleopteran insects but is quite variable in other insects. The determinants of efficient RNAi in coleopterans, as well as its potential mechanisms of resistance, are not known. RNAi screen identified a double-stranded RNA binding protein (StaufenC) as a major player in RNAi. StaufenC homologs have been identified in only coleopteran insects. Experiments in two coleopteran insects, and , showed the requirement of StaufenC for RNAi, especially for processing of double-stranded RNA (dsRNA) to small interfering RNA. RNAi-resistant cells were selected by exposing , Lepd-SL1 cells to the inhibitor of apoptosis 1 dsRNA for multiple generations. The resistant cells showed lower levels of StaufenC expression compared with its expression in susceptible cells. These studies showed that coleopteran-specific StaufenC is required for RNAi and is a potential target for RNAi resistance. The data included in this article will help improve RNAi in noncoleopteran insects and manage RNAi resistance in coleopteran insects.
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http://dx.doi.org/10.1073/pnas.1809381115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6099913PMC
August 2018

Recurrent triploidy of maternal origin.

Prenat Diagn 2000 Jul;20(7):561-3

Reproductive Genetics and Reproductive Endocrinology, Department of Obstetrics and Gynecology, Northwestern University Medical School, Chicago, IL, USA.

We report the occurrence of triploid preimplantation embryos following in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in a woman with two previously-identified triploid conceptuses which spontaneously underwent fetal demise at 10 and 23 weeks' gestation. An error in maternal meiosis II is proposed as the most likely cause.
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http://dx.doi.org/10.1002/1097-0223(200007)20:7<561::aid-pd875>3.0.co;2-1DOI Listing
July 2000