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Biopharmaceutical proteins are usually produced by culturing recombinant Chinese hamster ovary (CHO) cells. High producer cell lines are screened from transfected cells with random integration of target genes. Since transgene expression is susceptible to the surrounding environment of the integrated genomic locus, producer cell lines should be selected from a large number of recombinant cells with heterogeneous transgene insertion. In contrast, targeted integration into a characterized genomic locus allows for predictable transgene expression and less clonal variability, and thus stable production of target proteins can be expected. Genome editing technology based on programmable nucleases has recently emerged as a versatile tool for precise editing of target locus in the cell genome. Here, we demonstrated targeted knock-in of transgenes into the hypoxanthine phosphoribosyltransferase (hprt) locus of CHO cells using CRISPR/Cas9 and CRISPR-mediated precise integration into target chromosome (PITCh) systems. We also generated knock-in CHO cells based on the homology-independent targeted integration (HITI) system. We evaluated the knock-in efficiency of transgenes into the hprt locus using these systems.

Three-dimensional (3D) tissue construction from individual cells is an important process in regenerative medicine to enhance cell functions. In transplantation of tissue-engineered constructs, a limited oxygen/nutrient supply due to insufficient vascular network formation causes cell death. Thus, it is necessary to develop a system for inducing vascular networks into 3D tissue constructs under hypoxic conditions. In our previous study (Ono, A., et al., 2017), we developed a hypoxia-inducible transgene expression system in which a target gene can be expressed in response to hypoxic stress using hypoxia-responsive promoter RTP801 as a trigger, tTA transactivator as an amplifier, and oxygen-dependent degradation sequence as a noise canceler. In this study, to improve oxygen and nutritional limitation within engineered 3D tissue constructs, a hypoxia-responsive gene expression system for vascular endothelial growth factor (VEGF) was introduced into cells. We demonstrated that genetically engineered cells could regulate VEGF expression autonomously in an oxygen concentration-dependent manner. Using the genetically engineered cells, 3D tissue constructs were fabricated using a magnetic force-based tissue engineering technique (Ito, A., et al., 2005). The tissue constructs were transplanted into mice to evaluate the feasibility of the hypoxia-responsive VEGF gene expression system in vivo. The results indicated that the VEGF gene expression system is promising for the induction of vascular networks into 3D tissue constructs.

Hepatoma cells derived from liver carcinoma are a candidate cell source for bioartificial liver (BAL) systems due to their high proliferative capacity, although liver function of hepatoma cells is considerably low compared with primary hepatocytes. In our previous study, genetically engineered mouse hepatoma cells with inducible high liver function were established by transducing liver-enriched transcription factor (LETF) genes. In this study, we aimed to develop new gene-engineered human hepatoma cells, in which high liver functions are inducible by heat treatment. For this purpose, we constructed a gene expression system for eight LETF genes under control of tetracycline-dependent transactivator (tTA), and the system was introduced into the genome of HepG2-HSP cells, in which a tTA expression system induced by a heat-shock protein promoter with transcriptional amplification was introduced into HepG2 cells. Thus, the heat-inducible tTA promotes LETF genes to induce liver function. Upon the heat treatment of the cells (HepG2-HSP/8F) at 43°C for 30 min, liver functions such as albumin secretion and cytochrome P450 were significantly enhanced. The cells with heat-inducible liver function can be used as a new cell source for various hepatic studies including construction of BAL systems.

Gene delivery methods for animal cells are one of the most important tools in biotechnology fields such as pharmaceutical protein production, generation of transgenic animals and gene therapy. Because retrotransposons can move their own sequences to new genomic locations by a “copy-and-paste” process known as retrotransposition, we attempted to develop a novel gene transfer system based on retrotransposon. A full-length long interspersed element-1 (LINE-1) contains a 5’ untranslated region (5’UTR), two non-overlapping open reading frames (ORFs) separated by a short inter-ORF sequence, and a 3’UTR terminating in an adenosine-rich tract. We constructed a LINE-1 vector plasmid including components necessary for retrotransposition. An intron-disrupted Neo reporter gene and a scFv-Fc expression unit under the control of CMV promoter were added into 3’UTR in order to evaluate retrotransposition and express scFv-Fc. CHO-K1 cells transfected with the plasmids were screened with G418. The established cell clones produced scFv-Fc proteins in the culture medium. To control retrotransposition steadily, we also established retrotransposon systems that supply ORF2 or ORF1–2 separately. Genomic PCR analysis revealed that transgene sequences derived from the LINE-1 vector were positive in all clones. All the clones tested produced scFv-Fc in the culture medium.

Genetically modified microalgae have been expected to be a useful tool for bioenergy and recombinant protein production. However, random integration of transgene in the microalgae nuclear genome is susceptible to gene silencing of heterologous gene expression. Here, we attempted to perform targeted gene integration into a pre-determined nuclear genomic site of Chlamydomonas reinhardtii using Cre/loxP recombination system for stable transgene expression. We constructed an expression vector plasmid encoding reporter genes (zeocin resistant gene and green fluorescent protein gene; Zeo-2A-GFP) and mutated loxP to generate founder cells. A donor vector encoding IFNα-4 and paromomycin resistant genes flanked by corresponding mutated loxPs was constructed and introduced into founder cells together with a Cre expression vector. The optimal ratio of donor vector to Cre expression vector was determined by counting the number of paromomycin resistant colonies. For the established clones, the targeted integration was confirmed by genomic PCR using various specific primer sets. Target genes in the donor vector could be integrated into the expected genomic site of C. reinhardtii using Cre/loxP system. RT-PCR revealed that IFNα-4 was expressed in five independent transgenic cell lines tested. This result suggests that Cre-based cell engineering is a promising approach to generate smart microalgae expressing foreign genes.

Abstract An attempt was made to assign five genes, CPS1, OTC, ASS, CRYD2, and ARG2, to chicken chromosomes (GGA) by radiation-hybrid mapping. OTC was assigned to GGA1; ARG2 to GGA5; CPS1 to GGA7; and CRYD2 to GGA19. ASS was not, however, assigned to a specific chromosomal position.