Safety tests and clinical research on buccal and nasal microneedle swabs for genomic analysis.

Conventional swabs have been used as a non-invasive method to obtain samples for DNA analysis from the buccal and the nasal mucosa. However, swabs may not always collect pure enough genetic material. In this study, buccal and nasal microneedle swab is developed to improve the accuracy and reliability of genomic analysis. A cytotoxicity test, a skin sensitivity test, and a skin irritation test are conducted with microneedle swabs. Polymer microneedle swabs meet the safety requirements for clinical research and commercial use. When buccal and nasal microneedle swabs are used, the amount of genetic material obtained is greater than that from commercially available swabs, and DNA purity is also high. The comparatively short microneedle swab (250 µm long) cause almost no pain to all 25 participants. All participants also report that the microneedle swabs are very easy to use. When genotypes are compared at five SNP loci from blood of a participant and from that person's buccal or nasal microneedle swab, the buccal and nasal microneedle swabs show 100% concordance for all five SNP genotypes. Microneedle swabs can be effectively used for genomic analysis and prevention through genomic analysis, so the utilization of microneedle swabs is expected to be high.

Corrigendum: Development of a microneedle swab for acquisition of genomic DNA from buccal cells.

[This corrects the article DOI: 10.3389/fbioe.2022.829648.].

Development of a Microneedle Swab for Acquisition of Genomic DNA From Buccal Cells.

A swab is a tool for obtaining buccal DNA from buccal mucus for biological analysis. The acquisition of a sufficient amount and high quality of DNA is an important factor in determining the accuracy of a diagnosis. A microneedle swab (MN swab) was developed to obtain more oral mucosal tissues non-invasively. Eight types of MN swabs were prepared with varying combinations of patterns (zigzag or straight), number of MNs, intervals of MNs, and sharpness of tips. When MN swab was applied up to 10 times, the tissue amount and DNA yield increased compared to commercial swabs. A zigzag pattern of microneedles was found to be more efficient than a straight pattern and increasing the number of microneedles in an array increased the DNA yield. The MN swab collected about twice the DNA compared to the commercial swab. In an in vivo test using mini pigs, the lower cycle threshold values of mucosal samples collected with MN swabs compared to samples collected with commercial swabs indicated that a greater amount of DNA was collected for SNP genotyping. A polymer MN swab is easy to manufacture by a single molding process, and it has a greater sampling capacity than existing commercial swabs.

Secretory phospholipase A2-X (Pla2g10) is a novel progesterone receptor target gene exclusively induced in uterine luminal epithelium for uterine receptivity in mice.

BACKGROUND: Aberration of estrogen (E2) and/or progesterone (P4) signaling pathways affects expression of their target genes, which may lead to failure of embryo implantation and following pregnancy. Although many target genes of progesterone receptors (PRs) have been identified in uterine stroma, only a few PR targets have been reported in the epithelium. Secretory phospholipase A2-(PLA2)-X, a member of the PLA2 family that releases arachidonic acids for the synthesis of prostaglandins that are important for embryo implantation, is dysregulated in the endometrium of patients suffering from repeated implantation failure. However, it is not clear whether sPLA2-X is directly regulated by ovarian steroid hormones for embryo implantation in the uterus. RESULT: P4 induced the Pla2g10 encoding of secretory PLA2-X in the apical region of uterine LE of ovariectomized mice via PR in both time- and dose-dependent manners, whereas E2 significantly inhibited it. This finding is consistent with the higher expression of Pla2g10 at the diestrus stage, when P4 is elevated during the estrous cycle, and at P4-treated delayed implantation. The level of Pla2g10 on day 4 of pregnancy (day 4) was dramatically decreased on day 5, when PRs are absent in the LE. Luciferase assays of mutagenesis in uterine epithelial cells demonstrated that four putative PR response elements in a Pla2g10 promoter region are transcriptionally active for Pla2g10. Intrauterine delivery of small interfering RNA for Pla2g10 on day 3 significantly reduced the number of implantation sites, reinforcing the critical function(s) of Pla2g10 for uterine receptivity in mice. CONCLUSIONS: Pla2g10 is a novel PR target gene whose expression is exclusively localized in the apical region of the uterine LE for uterine receptivity for embryo implantation in mice.

Electroporation of AsCpf1/RNP at the Zygote Stage is an Efficient Genome Editing Method to Generate Knock-Out Mice Deficient in Leukemia Inhibitory Factor.

BACKROUND: CRISPR/Cpf1 is a class II, type V RNA-guided endonuclease that is distinct from the type II CRISPR/Cas9 nuclease, widely used for genome editing. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some limitations of the CRISPR/Cas9 system. The applications of CRISPR to rodent embryos for the production of knock-out (KO) mice have been achieved mainly by microinjection, which requires heavily-equipped instruments with skillful hands. Here, we evaluated the genome editing efficiency between Cpf1/mRNA and Cpf1/ribonuclear protein (RNP) in mouse embryos, and established an easy, fast, and technically less demanding method to produce KO mice using electroporation of the Cfp1/RNP system. METHODS: The efficiency of electroporation-based delivery of AsCpf1/mRNA and AsCpf1/RNP to target exon 3 of leukemia inhibitory factor (Lif) into mouse zygotes was evaluated. Embryos that developed to the two-cell stage after zygote electroporation were transferred into the oviducts of surrogate mothers to produce AsCpf1-mediated LIF KO mice. The genome editing efficiency of blastocysts and pups was tested using the T7E1 assay and/or DNA sequencing. Congenital abnormalities and reproductive phenotypes in LIF KO mice produced by electroporation with AsCpf1/RNP were examined. RESULTS: Survival and two-cell development of electroporated zygotes were comparable between the AsCpf1/mRNA and AsCpf1/RNP groups, whereas genome editing efficiency was relatively higher in the AsCpf1/RNP group (13.3% vs 18.1% at blastocyst and 33.3% vs 45.5% at offspring), respectively. Two mouse lines with a frameshift mutation in exon 3 of the Lif gene were established from the AsCpf1/RNP group. All congenital abnormalities of LIF KO mice produced by AsCpf1/RNP electroporation were observed. AsCpf1-mediated LIF KO mice showed postnatal growth retardation and implantation failure, both of which are major phenotypes of LIF KO mice generated by conventional gene targeting. CONCLUSION: Electroporation of AsCpf1/RNP at the zygote stage is an efficient genome editing method to produce KO mice.

Multifunctional nanoparticles for genetic engineering and bioimaging of natural killer (NK) cell therapeutics.

Recently, natural killer (NK)-based immunotherapy has attracted attention as a next-generation cell-based cancer treatment strategy due to its mild side effects and excellent therapeutic efficacy. Here, we describe multifunctional nanoparticles (MF-NPs) capable of genetically manipulating NK cells and tracking them in vivo through non-invasive magnetic resonance (MR) and fluorescence optical imaging. The MF-NPs were synthesized with a core-shell structure by conjugation of a cationic polymer labeled with a near-infrared (NIR) fluorescent molecule, with the aid of a polydopamine (PDA) coating layer. When administered to NKs, the MF-NPs exhibited excellent cytocompatibility, efficiently delivered genetic materials into the immune cells, and induced target protein expression. In particular, the MF-NPs could induce the expression of EGFR targeting chimeric antigen receptors (EGFR-CARs) on the NK cell surface, which improved the cells' anti-cancer cytotoxic effect both in vitro and in vivo. Finally, when NK cells labeled with MF-NPs were injected into live mice, MF-NP-labeled NK cells could be successfully imaged using fluorescence and MR imaging devices. Our findings indicate that MF-NPs have great potential for application of NK cells, as well as other types of cell therapies involving genetic engineering and in vivo monitoring of cell trafficking.