Monoclonal Cell Lines Research Articles

Chorioallantoic Membrane Tumor Model for Evaluating Oncolytic Viruses.

Oncolytic viruses are promising anticancer agents; however, regarding their clinical efficacy, there is still significant scope for improvement. Preclinical in vivo evaluation of oncolytic viruses is mainly based on syngeneic or xenograft tumor models in mice, which is labor-intensive and time-consuming. Currently, a large proportion of developmental work in the research field of oncolytic viruses is directed toward overcoming cellular and noncellular barriers to achieve improved virus delivery to primary tumors and metastases. To evaluate the large number of genetically or chemically modified viruses regarding tumor delivery and biodistribution patterns, it would be valuable to have an in vivo model available that would allow easy screening experiments, that is of higher complexity than monoclonal cell lines, and that could be used as a platform method before confirmatory studies in small and large animals. Based on our data, we believe that the chicken chorioallantoic membrane (CAM) assay is a quick and low-cost high-throughput tumor model system for the in vivo analysis of oncolytic viruses. Here we describe the establishment, careful characterization, and optimization of the CAM model as an in vivo model for the evaluation of oncolytic viruses. We have used human adenovirus type 5 (HAdV-5) as an example for validation but are confident that the model can be used as a test system for replicating viruses of many different virus families. We show that the CAM tumor model enables intratumoral and intravenous virus administration and is a feasible and conclusive model for the analysis of relevant virus–host interactions, biodistribution patterns, and tumor-targeting profiles.

Research on the effect of PD-L1 overexpression on CLL-1 CAR-T anti-acute myeloid leukemia

目的探讨程序性死亡受体配体1（PD-L1）对CLL-1 CAR-T细胞抗急性髓系白血病（AML）作用的影响。方法通过构建PD-L1表达载体、制备慢病毒、转导、单克隆筛选技术获得稳定表达PD-L1的THP-1单克隆细胞株（THP1-PDL1），然后以前期制备的CLL-1 CAR-T细胞为效应细胞，以THP-1、THP1-PDL1单克隆细胞株作为靶细胞，分别通过LDH检测、CBA法、CFSE法评价PD-L1过表达对CLL-1 CAR-T细胞裂解功能、炎性因子释放、细胞增殖等功能的影响。结果①成功制备了PD-L1慢病毒，并筛选获得了稳定表达PD-L1的THP1-PDL1单克隆细胞株，流式细胞术及PCR验证成功。②PD-L1过表达抑制了CLL-1 CAR-T细胞裂解THP-1细胞的能力；效靶比为10∶1时，CLL-1CAR-T细胞对THP1-PDL1细胞的杀伤效率明显低于对THP-1细胞的杀伤效率［（15.70±9.90）％对（51.95±2.52）％，P<0.05］。③PD-L1过表达减弱了CLL-1 CAR-T细胞释放细胞因子的能力［与THP1-PDL1细胞共培养时对与THP-1细胞共培养时：IFN-γ（115.66±3.13）pg/ml对（1708.16±26.76）pg/ml，P<0.05；IL-6（17.37±0.72）pg/ml对（124.92±4.26）pg/ml，P<0.05；IL-10（5.69±0.13）pg/ml对（124.12±3.02）pg/ml，P<0.05］；同时抑制了CLL-1 CAR-T细胞的增殖能力。结论成功构建了表达PD-L1的THP1-PDL1单克隆细胞株，同时证实了PD-L1过表达对CLL-1 CAR-T细胞抗AML的不利的影响，为通过PD-1/PD-L1通路调控CLL-1 CAR-T细胞功能提供了一定的理论基础。

Establishment of a low-tumorigenic MDCK cell line and study of differential molecular networks

Influenza is an acute respiratory infection caused by the influenza virus, and vaccination against influenza is considered the best way to prevent the onset and spread. MDCK (Madin-Darby canine kidney) cells are typically used to isolate the influenza virus, however, their high tumorigenicity is the main controversy in the production of influenza vaccines. Here, MDCK-C09 and MDCK-C35 monoclonal cell lines were established, which were proven to be low in tumorigenicity. RNA-seq of MDCK-C09, MDCK-C35, and MDCK-W73 cells was performed to investigate the putative tumorigenicity mechanisms. Tumor-related molecular interaction analysis of the differentially expressed genes indicates that hub genes, such as CUL3 and EGFR, may play essential roles in tumorigenicity differences between MDCK-C (MDCK-C09 and MDCK-C35) and MDCK-W (MDCK-W73) cells. Moreover, the analysis of cell proliferation regulation-associated molecular interaction shows that downregulated JUN and MYC, for instance, mediate increased proliferation of these cells. The present study provides a new low-tumorigenic MDCK cell line and describes the potential molecular mechanism for the low tumorigenicity and high proliferation rate.

Generation and characterization of CRISPR/Cas9-mediated MEN1 knockout BON1 cells: a human pancreatic neuroendocrine cell line

Among patients with the rare diagnosis of pancreatic neuroendocrine tumor (P-NET), a substantial proportion suffer from the inherited cancer syndrome multiple endocrine neoplasia type 1 (MEN1), which is caused by germline mutations of the MEN1 suppressor gene. Somatic mutations and loss of the MEN1 protein (menin) are frequently also found in sporadic P-NETs. Thus, a human neuroendocrine pancreatic cell line with biallelic inactivation of MEN1 might be of value for studying tumorigenesis. We used the polyclonal human P-NET cell line BON1, which expresses menin, serotonin, chromogranin A and neurotensin, to generate a monoclonal stable MEN1 knockout BON1 cell line (MEN1-KO-BON1) by CRISPR/Cas9 editing. Changes in morphology, hormone secretion, and proliferation were analyzed, and proteomics were assessed using nanoLC-MS/MS and Ingenuity Pathway Analysis (IPA). The menin-lacking MEN1-KO-BON1 cells had increased chromogranin A production and were smaller, more homogenous, rounder and grew faster than their control counterparts. Proteomic analysis revealed 457 significantly altered proteins, and IPA identified biological functions related to cancer, e.g., posttranslational modification and cell death/survival. Among 39 proteins with at least a two-fold difference in expression, twelve are relevant in glucose homeostasis and insulin resistance. The stable monoclonal MEN1-KO-BON1 cell line was found to have preserved neuroendocrine differentiation, increased proliferation, and an altered protein profile.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Development of a cell-based diagnostic system for vitamin K-dependent coagulation factor deficiency 1

To develop a cell-based system for the diagnosis of vitamin K-dependent coagulation factor deficiency 1 (VKCFD1). In HEK293 cells stably expressing the reporter gene FIX-Gla-PC, the gamma-glutamyl carboxylase (GGCX) gene was knocked out by using CRISPR/Cas9 technology. Enzyme-linked immunosorbent assay (ELISA), DNA sequencing and Western blotting were used to identify the GGCX gene knockout cells. A quickchange point variant method was used to construct the GGCX variant. ELISA was used to assess the influence of GGCX variant on the activity of reporter gene. Two monoclonal cell lines with no reporter activity by ELISA was identified. Edition and knockout of the GGCX gene was confirmed by DNA sequencing and Western blotting. The activity of the reporter gene was recovered by transfection of the wild-type GGCX gene. Thereby two monoclonal cells with GGCX knockout were obtained. By comparing the wild-type and pathogenic GGCX variants, the reporter activity was decreased in the pathogenic variants significantly. A cell-based system for the detection of GGCX activity was successfully developed, which can be used for the diagnosis of VKCFD1 caused by GGCX variants.

One Cell, One Drop, One Click: Hybrid Microfluidics for Mammalian Single Cell Isolation

Generating a stable knockout cell line is a complex process that can take several months to complete. In this work, a microfluidic method that is capable of isolating single cells in droplets, selecting successful edited clones, and expansion of these isoclones is introduced. Using a hybrid microfluidics method, droplets in channels can be individually addressed using a co-planar electrode system. In the hybrid microfluidics device, it is shown that single cells can be trapped and subsequently encapsulate them on demand into pL-sized droplets. Furthermore, droplets containing single cells are either released, kept in the traps, or merged with other droplets by the application of an electric potential to the electrodes that is actuated through an in-house user interface. This high precision control is used to successfully sort and recover single isoclones to establish monoclonal cell lines, which is demonstrated with a heterozygous NCI-H1299 lung squamous cell population resulting from loss-of-function eGFP and RAF1 gene knockout transfections.

P14ARF: The Absence that Makes the Difference.

P14ARF is a tumor suppressor encoded by the CDKN2a locus that is frequently inactivated in human tumors. P14ARF protein quenches oncogene stimuli by inhibiting cell cycle progression and inducing apoptosis. P14ARF functions can be played through interactions with several proteins. However, the majority of its activities are notoriously mediated by the p53 protein. Interestingly, recent studies suggest a new role of p14ARF in the maintenance of chromosome stability. Here, we deepened this new facet of p14ARF which we believe is relevant to its tumor suppressive role in the cell. To this aim, we generated a monoclonal HCT116 cell line expressing the p14ARF cDNA cloned in the piggyback vector and then induced aneuploidy by treating HCT116 cells with the CENP-E inhibitor GSK923295. P14ARF ectopic re-expression restored the near-diploid phenotype of HCT116 cells, confirming that p14ARF counteracts aneuploid cell generation/proliferation.

A Microfluidic Single-Cell Cloning (SCC) Device for the Generation of Monoclonal Cells.

Single-cell cloning (SCC) is a critical step in generating monoclonal cell lines, which are widely used as in vitro models and for producing proteins with high reproducibility for research and the production of therapeutic drugs. In monoclonal cell line generation, the development time can be shortened by validating the monoclonality of the cloned cells. However, the validation process currently requires specialized equipment that is not readily available in general biology laboratories. Here, we report a disposable SCC device, in which single cells can be isolated, validated, and expanded to form monoclonal cell colonies using conventional micropipettes and microscopes. The monoclonal cells can be selectively transferred from the SCC chip to conventional culture plates, using a tissue puncher. Using the device, we demonstrated that monoclonal colonies of actin-GFP (green fluorescent protein) plasmid-transfected A549 cells could be formed in the device within nine days and subsequently transferred to wells in plates for further expansion. This approach offers a cost-effective alternative to the use of specialized equipment for monoclonal cell generation.

Expression analysis of tPA/gGH double genes in transfected goat mammary epithelial cells

tPA is a thrombolytic agent widely used in clinical settings. While double gene co-integration into organisms can produce synergistic effects and improved expression levels of the target gene, there are few reports detailing the co-integration of the tPA and gGH genes and an increased expression level of tPA. In order to study this, we obtained monoclonal goat mammary epithelial cell lines with tPA/gGH double gene integration and we analyzed the tPA expression level of single and double gene integration cells. We constructed a mammary gland-specific expression vector PCL25/gGH by using the β-casein gene as the regulatory sequence. The tPA and gGH genes were co-transfected into goat mammary epithelial cells by electrotransfection. Resistant cell lines were screened by G418, and transgenic monoclonal cell lines were obtained by PCR detection. tPA expression was induced by prolactin and subsequently, the cell induction solution was assayed after 48 hours by ELISA and Western blotting. The results show that a total of 142 resistant monoclonal cells were obtained including 53 tPA monogenic integration cell lines and 34 tPA/gGH double gene integration cell lines. The rate of double gene integration was 23.9% (34/142). A total of 29 cells were detected to be able to express tPA, of which 12 were single-gene-expressing cells and the corresponding expression rate was 22.6% (12/53). There were 17 double-gene- expressing cells with a corresponding expression rate of 50.0% (17/34). The expression level of tPA in single-gene cells was 7.5-52.0 μg/mL, while in double-gene cells was 40-360 μg/mL, which was significantly greater than that in single- gene cells. The goat mammary epithelial cell lines with tPA/gGH gene integration were successfully obtained by electrotransfection, and we proved that the expression level of tPA in the double gene integration cell lines with tPA/gGH gene integration was significantly increased. Our findings lay the foundation for the additional study of highly expressed transgenic goats and other animals with determination of scientific and clinical utility.

A porcine alveolar macrophage cell line stably expressing CD163 demonstrates virus replication and cytokine secretion characteristics similar to primary alveolar macrophages following PRRSV infection

The in vitro investigation of cytokine secretion induced by porcine reproductive and respiratory syndrome virus (PRRSV) requires porcine alveolar macrophages (PAMs) and their interaction with immunocytes. However, immortalized monoclonal PAMs (mPAMs) are non-permissive for PRRSV infection. The porcine CD163 receptor isolated from primary PAMs (pPAMs) confers susceptibility to PRRSV infection; thus, this approach could be used to establish a novel cell line to facilitate the exploration of PRRSV infection kinetics. Here, we amplified the coding region of the CD163 gene from pPAMs and integrated it into an mPAM line using a lentivirus expression system. After verification, the monoclonal PAM cell line stably expressing CD163 (mPAM-CD163-GFP) was infected with either the highly pathogenic PRRSV strain JXA1 or the classical PRRSV strain SD1, which produced high infectious titers of progeny virus reaching > 109 copies/mL or a 50 % tissue culture infective dose of 105.5 over at least 100 cell generations. We also investigated cytokine and Toll-like receptor expression in infected mPAM-CD163-GFP cells and pPAMs. The mPAM-CD163-GFP cell line showed similar patterns of viral replication and cytokine secretion compared with pPAMs, so it may be extremely useful for replacing primary cells for in vitro investigations of the mechanisms of cytokine secretion and interactions between PRRSV-infected PAMs and immunocytes.

Improvement of Intracellular Traffic System by Overexpression of KDEL Receptor 1 in Antibody-Producing CHO Cells.

The localization of soluble endoplasmic reticulum (ER) chaperones in the cell organelle is mediated by the C-terminal KDEL (lysine, aspartic acid, glutamic acid and leucine) motif. This motif is recognized by the KDEL receptor, a seven-transmembrane protein that cycles between the ER and cis-Golgi to capture missorted KDEL chaperones from post-ER compartments in a pH-dependent manner. The KDEL receptor's target chaperones have a substantial role in protein folding and assembly. In this study, the gene expression level of KDEL receptor 1 shows a moderate upregulation during either ER stress or growth of Chinese hamster ovary (CHO) cells in batch culture, while the ER chaperones show higher upregulation. This might indicate the possibility of saturation of the ER retention machinery or at least hindered retention during late stage batch culture in recombinant CHO cells. KDELR1 is overexpressed in a monoclonal antibody-producing CHO cell line to improve the intracellular chaperone retention rate in the ER. An increase in the specific productivity of IgG1 by 13.2% during the exponential phase, and 23.8% in the deceleration phase of batch culture is observed. This is the first study to focus on the ER retention system as a cell engineering target for enhancing recombinant protein production.

BGC823 Cell Line with the Stable Expression of iRFP720 Retains Its Primary Properties with Promising Fluorescence Imaging Ability.

Reliable animal models are required for understanding the molecular events of gastric tumor growth and metastasis. Tracing techniques based on iRFP720 may optimize the noninvasive monitoring of tumors in vivo. The present study established a human gastric adenocarcinoma cell line BGC823-iRFP720-GFP (abbreviated as BGC823-iRFP) that stably expressed iRFP720 and green fluorescent protein (GFP) by piggyBac transposon system. The monoclonal cell line BGC823-iRFP was isolated under puromycin selection. The cell morphology and proliferation ability of BGC823-iRFP cells in vitro were similar to that of the BGC823 cells. The iRFP720 and GFP expressions were confirmed by laser confocal microscopy and Cytation™ 5. Hematoxylin and eosin staining, immunohistochemical analysis, and animal experiments also revealed that BGC823-iRFP exhibited no significant changes in morphology, growth kinetics, and tumorigenicity in vivo. IVIS Lumina III imaging indicated that the iRFP720 signals of the BGC823-iRFP cells could be used to evaluate the antitumor efficacy of oncolytic viruses and chemotherapy drugs. Therefore, the BGC823-iRFP cells would be a useful tool for gastric cancer research and antitumor drug evaluation.

Characterisation of endogenous Claudin-1 expression, motility and susceptibility to hepatitis C virus in CRISPR knock-in cells.

Claudin-1 (CLDN1) is a four-span transmembrane protein localised at cell-cell tight junctions (TJs), playing an important role in epithelial impermeability and tissue homoeostasis under physiological conditions. Moreover, CLDN1 expression is up-regulated in several cancers, and the level of CLDN1 expression has been proposed as a prognostic marker of patient survival. Here, we generated and characterised a novel reporter cell line expressing endogenous fluorescent levels of CLDN-1, allowing dynamic monitoring of CLDN-1 expression levels. Specifically, a hepatocellular carcinoma Huh7.5.1 monoclonal cell line was bioengineered using CRISPR/Cas9 to endogenously express a fluorescent TagRFP-T protein fused at the N-terminus of the CLDN1 protein. These cells were proved useful to measure CLDN1 expression and distribution in live cells. However, the cells were resistant to hepatitis C virus (HCV) infection, of which CLDN1 is a viral receptor, while retaining permissiveness to VSV-G-decorated pseudoparticles. Nonetheless, the TagRFP-CLDN1+/+ cell line showed expected CLDN1 protein localisation at TJs and the cell monolayer had similar impermeability and polarisation features as its wild-type counterpart. Finally, using fluorescence recovery after photobleaching (FRAP) approaches, we measured that the majority of endogenous and overexpressed TagRFP-CLDN1 diffuses rapidly within the TJ, whereas half of the overexpressed EGFP-CLDN1 proteins were stalled at TJs. The Huh7.5.1 TagRFP-CLDN1+/+ edited cell line showed physiological features comparable to that of non-edited cells, but became resistant to HCV infection. Our data also highlight the important impact of the fluorescent protein chosen for endogenous tagging. Although HCV-related studies may not be achieved with these cells, our work provides a novel tool to study the cell biology of TJ-associated proteins and a potential screening strategy measuring CLDN1 expression levels.

Detecting heterogeneity in and between breast cancer cell lines

BackgroundCellular heterogeneity in tumor cells is a well-established phenomenon. Genetic and phenotypic cell-to-cell variability have been observed in numerous studies both within the same type of cancer cells and across different types of cancers. Another known fact for metastatic tumor cells is that they tend to be softer than their normal or non-metastatic counterparts. However, the heterogeneity of mechanical properties in tumor cells are not widely studied.ResultsHere we analyzed single-cell optical stretcher data with machine learning algorithms on three different breast tumor cell lines and show that similar heterogeneity can also be seen in mechanical properties of cells both within and between breast tumor cell lines. We identified two clusters within MDA-MB-231 cells, with cells in one cluster being softer than in the other. In addition, we show that MDA-MB-231 cells and MDA-MB-436 cells which are both epithelial breast cancer cell lines with a mesenchymal-like phenotype derived from metastatic cancers are mechanically more different from each other than from non-malignant epithelial MCF-10A cells.ConclusionSince stiffness of tumor cells can be an indicator of metastatic potential, this result suggests that metastatic abilities could vary within the same monoclonal tumor cell line.

DAPK1 loss triggers tumor invasion in colorectal tumor cells

Colorectal cancer (CRC) is one of the leading cancer-related causes of death worldwide. Despite the improvement of surgical and chemotherapeutic treatments, as of yet, the disease has not been overcome due to metastasis to distant organs. Hence, it is of great relevance to understand the mechanisms responsible for metastasis initiation and progression and to identify novel metastatic markers for a higher chance of preventing the metastatic disease. The Death-associated protein kinase 1 (DAPK1), recently, has been shown to be a potential candidate for regulating metastasis in CRC. Hence, the aim of the study was to investigate the impact of DAPK1 protein on CRC aggressiveness. Using CRISPR/Cas9 technology, we generated DAPK1-deficient HCT116 monoclonal cell lines and characterized their knockout phenotype in vitro and in vivo. We show that loss of DAPK1 implemented changes in growth pattern and enhanced tumor budding in vivo in the chorioallantoic membrane (CAM) model. Further, we observed more tumor cell dissemination into chicken embryo organs and increased invasion capacity using rat brain 3D in vitro model. The novel identified DAPK1-loss gene expression signature showed a stroma typical pattern and was associated with a gained ability for remodeling the extracellular matrix. Finally, we suggest the DAPK1-ERK1 signaling axis being involved in metastatic progression of CRC. Our results highlight DAPK1 as an anti-metastatic player in CRC and suggest DAPK1 as a potential predictive biomarker for this cancer type.

Are Small Nucleolar RNAs "CRISPRable"? A Report on Box C/D Small Nucleolar RNA Editing in Human Cells.

CRISPR technologies are nowadays widely used for targeted knockout of numerous protein-coding genes and for the study of various processes and metabolic pathways in human cells. Most attention in the genome editing field is now focused on the cleavage of protein-coding genes or genes encoding long non-coding RNAs (lncRNAs), while the studies on targeted knockout of intron-encoded regulatory RNAs are sparse. Small nucleolar RNAs (snoRNAs) present a class of non-coding RNAs encoded within the introns of various host genes and involved in post-transcriptional maturation of ribosomal RNAs (rRNAs) in eukaryotic cells. Box C/D snoRNAs direct 2’-O-methylation of rRNA nucleotides. These short RNAs have specific elements in their structure, namely, boxes C and D, and a target-recognizing region. Here, we present the study devoted to CRISPR/Cas9-mediated editing of box C/D snoRNA genes in Gas5. We obtained monoclonal cell lines carrying mutations in snoRNA genes and analyzed the levels of the mutant box C/D snoRNA as well as the 2’-O-methylation status of the target rRNA nucleotide in the obtained cells. Mutations in SNORD75 in the obtained monoclonal cell line were shown to result in aberrant splicing of Gas5 with exclusion of exons 3 to 5, which was confirmed by RT-PCR and RNA-Seq. The obtained results suggest that SNORD75 contains an element for binding of some factors regulating maturation of Gas5 pre-lncRNA. We suggest that METTL3/METTL14 is among such factors, and m6A-methylation pathways are involved in regulation of Gas5 splicing. Our results shell light on the role of SNORDs in regulating splicing of the host gene.

The Function of E6BP in Normal Human and HPV-Infected Cell Lines

Background and Hypothesis: High-risk human papillomavirus type-16 and -18 cause approximately 70% of cervical and 60% of oropharyngeal cancers. While the majority of HPV infections are transient and resolve on their own, some infections persist and progress to cancer. The in vivo and in vitro high-risk HPV transformation ability works to immortalize primary human keratinocytes resulting from the activities of the viral oncoproteins E6 and E7. A well-studied effect of E6 is the degradation of tumor suppressor gene p53 via binding to the ubiquitin ligase E6AP. Additionally, E6 interacts with the cellular calcium-binding protein E6BP, but the functional significance remains unknown. Here we studied the effects of E6BP knockout in normal human and HPV-infected cell lines.&#x0D; Experimental Design or Project Methods: CRISPR-Cas9 system was used to knockout E6BP in HPV-human cervical cancer cell lines: HeLa (high-copy HPV18) and SiHa (low-copy HPV16) as well as human keratinocyte HaCaT (mutant p53) and p53-null lung carcinoma H1299 cell lines. Cells were transfected with a puromycin-resistant CRISPR*Cas9 control plasmid (pLentiv2) and one that carries a guide RNA against E6BP (pLentiv2-E6BP) and selected with puromycin. RFP-GFP, which does not carry a puromycin resistance, was transfected into cells to establish transfection efficiency and to serve as the control for the puromycin. After successful selection, polyclonal populations of pLentiv2 control and pLentiv2-E6BP cells were screened for E6BP protein levels. pLentiv2-E6BP cell populations with reduced E6BP protein were serial diluted to generate monoclonal cell lines. Clones were then screened E6BP protein expression, and low expression clones were further processed for DNA sequencing of the E6BP gene editing site. Additionally, the effects of E6BP knockout in Siha and HeLa cells on p53, p21, and E6AP protein expression was studied by immunoblot.&#x0D; Results: Monoclonal pLentiv2-E6BP cell lines for H1299 and HaCaT cells were established and as well as polyclonal pLentiv2-E6BP HeLa and SiHa cell populations. Preliminary results indicate that E6BP knockout in HeLa and Siha cells reduces the p53 and p21 levels compared to pLentiv2 control cells. Additionally, E6AP protein expression was not affected by E6BP knockout in the polyclonal SiHa cells.&#x0D; Conclusion and Potential Impact: These preliminary results suggest that E6BP levels may influence p53 and p21 levels in HPV cells. It is of particular importance to further investigate if low levels of E6BP contribute to a worse outcome during HPV induced carcinogenesis.&#x0D;

Nanofountain Probe Electroporation for Monoclonal Cell Line Generation.

In the field of genetic engineering, the modification of genes to produce stable cell lines has a variety of applications ranging from the development of novel therapeutics to patient specific treatments. To successfully generate a cell line, the gene of interest must be delivered into the cell and integrated into the genome. The efficiency of cell line generation systems therefore depends on the efficiency of delivery of genetically modifying molecules such as plasmids and CRISPR/CAS9 complexes. In this work, we describe a localized electroporation-based system to generate stable monoclonal cell lines. By employing the nanofountain probe electroporation (NFP-E) system, single cells in patterned cultures are selectively transfected with plasmids, grown, and harvested to obtain stably expressing cell lines. Methods for microcontact printing, cell culture, electroporation, and harvesting are detailed in this chapter.

Preparation of an anti-isoprocarb monoclonal antibody and its application in developing an immunochromatographic strip assay.

In this study, a carboxyl group was introduced into the isoprocarb molecule to obtain an isoprocarb hapten, which was then coupled with a protein to obtain an artificial antigen. Three monoclonal antibody cell lines, 1D11, 6E6 and 1B5, were finally obtained by mouse immunization, cell fusion and subcloning, and the antibody produced by cell line 1B5 had the best affinity and sensitivity. The monoclonal antibody was highly sensitive and specific for isoprocarb, with an IC50 of 2.09 ng/ml and a cross-reactivity rate of <0.21%. By optimizing the indirect competitive (ic)-ELISA, the optimal conditions were determined to be pH 7.4, 0% methanol and 0.8% NaCl, the limit of detection value was 0.23 ng/ml, and the linear range of the ic-ELISA was 0.46-9.62 ng/ml. The recovery rate of the isoprocarb cucumber sample was 97-99% for the ic-ELISA method. In addition, we successfully developed an immunochromatographic test strip for the detection of isoprocarb residues. The cutoff values in phosphate-buffered saline and cucumber extract were 10 and 25 ng/ml, respectively. Both methods met the requirements for isoprocarb residue detection in agricultural products, and can be used for semiquantitative and qualitative analysis of isoprocarb in vegetables.