Removal Of Selectable Markers Research Articles

A novel screening system based on gene targeting to enrich the modified mammalian cells: without leaving selection marker and additional sequence

Gene targeting by homologous recombination (HR) has some disadvantages in screening modified cells that limits their use in targeting gene fragments in long exons. These disadvantages include retention of remaining selection marker after targeting, not removing cells with vector random integration, and leaving loxP sequences after removal of selection markers. Therefore, to overcome these disadvantages, we decided to design a eukaryotic two-step screening system to isolate the favorable, edited cells from undesirable cells in a gene targeting project. This system included two targeting plasmids containing one positive marker and two inducible negative markers. It was designed in such a way that, during the two-step HR and subsequent selection, only the well-edited cells survive and cells with vector random integration, and untargeted and episomal targeting plasmids are eliminated. The percentage of GFP-positive cells in two-step screening method (76.10 ± 3.50) was significantly higher than in the one-step screening method (0.90 ± 0.37) (p < 0.0001). GFP noise caused by the presence of the GFP-episomal expression plasmid had no significant effect on our results. We developed an efficient system to screen and enrich the HR-modified cells from undesired-HR and untargeted cells, without leaving the selection markers in mammalian cells. This method may be a promising method in ex vivo gene therapy approaches, especially when the target is a gene fragment within a large exon.

Abstract 2978: The utilization of a human MCL1 knockin mouse suggests that reductions in B-cells and monocytes may serve as clinically relevant pharmacodynamic markers of MCL1 inhibition

Abstract The development of small-molecule inhibitors targeting pro-survival BCL-2 family members, such as MCL1 and BCL-2, is an attractive approach for the treatment of cancer. AMG 176 is a potent and selective first-in-class MCL1 inhibitor currently in phase I clinical trials in multiple myeloma and AML. AMG 176 and its analogue AM-8621 exhibit picomolar affinity for human MCL1. However, binding affinity for murine MCL1 is reduced by ~200-fold. Examination of the AMG 176 binding pocket in human MCL1 suggests Leu248 contributes to the high affinity interaction with AMG 176. In mouse MCL1, the corresponding amino acid is a phenylalanine, reducing the affinity of AMG 176 for the mouse protein. This reduced affinity makes it challenging to assess the pharmacodynamic effect of AMG 176 on normal hematopoietic cell types at efficacious doses in human tumor xenograft models. To overcome this challenge we generated a human MCL1 knockin mouse in which the mouse genomic Mcl1 locus was replaced with its human counterpart. Human MCL1 knockin (KI) mice were created by targeting embryonic stem (ES) cells with a vector containing the human MCL1 genomic locus flanked by the homologous mouse sequences up- and downstream of mouse Mcl1. Targeted ES cells were confirmed by Southern blot and used to generate chimeric mice. Germline transmission of the human MCL1 KI allele and the removal of selectable markers was accomplished by breeding with cre-expressing transgenic mice. Homozygous human MCL1 KI mice were created by breeding heterozygous human MCL1 KI mice lacking the Cre transgene and selectable markers. We used a splenocyte viability assay to confirm the functional replacement of murine Mcl1 with its human ortholog. The in vitro treatment of splenocytes with AM-8621 for 6 hours resulted in dose-dependent Caspase 3 activation and reduced viability in B and T cells derived from human MCL1 knockin mice. Oral administration of AMG 176 at doses of 30 and 60 mg/kg resulted in the induction of apoptosis and the subsequent inhibition and regression of OPM-2 multiple myeloma tumor xenografts (54% tumor growth inhibition and 21% regression, respectively). The human MCL1 knockin mice were used to test the effect of AMG 176 at an equivalent dose and schedule on B cells, T cells and monocytes derived from peripheral blood, spleen and bone marrow. Samples treated with 30 and 60 mg/kg of AMG 176 were harvested 24 hours post-second dose of the first and second cycles. We observed a dose-dependent decrease in monocytes and B cells at both time points, with the most significant reductions observed in monocytes (70.1% and 94.5%, respectively) and B cells (81.5% and 94.0%, respectively) derived from peripheral blood after the first cycle. In conclusion, these data suggest that reductions in B cells and monocytes may serve as clinically relevant pharmacodynamic endpoints of MCL1 inhibition. Citation Format: Brian Belmontes, Sean R. Caenepeel, Jan Sun, Danny Chui, Angela Coxon, Gordon Moody, Jude Canon, Paul Hughes. The utilization of a human MCL1 knockin mouse suggests that reductions in B-cells and monocytes may serve as clinically relevant pharmacodynamic markers of MCL1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2978.

Genome editing through large insertion leads to the skipping of targeted exon.

BackgroundHighly efficient genome editing can be achieved through targeting an endonuclease to specific locus of interest. Engineered zinc-finger nuclease (ZFN) and CRISPR-associated protein-9 nuclease (Cas9) offer such an elegant approach for genome editing in vertebrate cells. In this study, we have utilized ZFN and Cas9-catalyzed double strand break followed by homologous recombination-mediated incorporation of premature stop codon and selection marker to target human cell division cycle 14A (hCDC14A) and cell division cycle 14B (hCDC14B) genes.ResultsTargeting of the hCDC14A and hCDC14B loci in telomerase immortalized human retinal pigment epithelium (hTERT-RPE1) and human colon cancer (HCT116) cells were confirmed by Southern blot hybridization. Nevertheless, DNA sequence analysis of reverse transcription polymerase chain reaction (RT-PCR) products confirmed that in all the single/double allele ablations, the targeted exon was spliced out. The phenomenon of exon skipping was independent of the genome editing approaches exploited, Cas9 or ZFN. Because the exons had a nucleotide number that could be divided by 3, the reading frame of the exon deletion was maintained. This indicates an exon-skipping event possibly due to the insertion of large DNA fragment (1.7 to 2.5 Kb) within the targeted exons. As a proof-of-principle, we have used gene disruption followed by non-homologous end joining (NHEJ) approach. Small alterations in the exon (one to fifteen bases) were transcribed to mRNA without exon skipping. Furthermore, loxP site-mediated removal of selection markers left a 45 bp scar within the targeted exon that can be traced in mRNA without exon skipping.ConclusionFrom this study, we conclude that insertion of a large DNA fragment into an exon by genome editing can lead to its skipping from the final transcript. Hence, more cautious approach needs to be taken while designing target sites in such that the possible skipping of targeted exon causes a frame-shift mediated incorporation of pre-mature stop codon. On the other hand, exon skipping may be a useful strategy for the introduction of protein deletions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2284-8) contains supplementary material, which is available to authorized users.

Conditional knockout of Foxc2 gene in kidney: efficient generation of conditional alleles of single-exon gene by double-selection system

Foxc2 is a single-exon gene and a key regulator in development of multiple organs, including kidney. To avoid embryonic lethality of conventional Foxc2 knockout mice, we conditionally deleted Foxc2 in kidneys. Conditional targeting of a single-exon gene involves the large floxed gene segment spanning from promoter region to coding region to avoid functional disruption of the gene by the insertion of a loxP site. Therefore, in ES cell clones surviving a conventional single-selection, e.g., neomycin-resistant gene (neo) alone, homologous recombination between the long floxed segment and target genome results in a high incidence of having only one loxP site adjacent to the selection marker. To avoid this limitation, we employed a double-selection system. We generated a Foxc2 targeting construct in which a floxed segment contained 4.6kb mouse genome and two different selection marker genes, zeocin-resistant gene and neo, that were placed adjacent to each loxP site. After double-selection by zeocin and neomycin, 72 surviving clones were screened that yielded three correctly targeted clones. After floxed Foxc2 mice were generated by tetraploid complementation, we removed the two selection marker genes by a simultaneous-single microinjection of expression vectors for Dre and Flp recombinases into in vitro-fertilized eggs. To delete Foxc2 in mouse kidneys, floxed Foxc2 mice were mated with Pax2-Cre mice. Newborn Pax2-Cre; Foxc2(loxP/loxP) mice showed kidney hypoplasia and glomerular cysts. These results indicate the feasibility of generating floxed Foxc2 mice by double-selection system and simultaneous removal of selection markers with a single microinjection.

Conditional removal of selectable markers in Trypanosoma cruzi using a site-specific recombination tool: Proof of concept

The scarcity of molecular tools for genetic manipulation is a critical obstacle for functional genomics studies on Trypanosoma cruzi. The current study adapted an inducible site-specific recombination system based on Dimerizable CRE recombinase (DiCRE). Two vectors for stable transfection were created, a first one to express inactive portions of DiCRE recombinase, and a second plasmid containing the loxP sites to test DiCRE activity. After integrating both constructs into the T. cruzi genome, it was shown that DiCRE recombinase can be efficiently used to manipulate its genome by allowing the removal of selectable markers thus generating homogeneous populations. The DiCRE recombinase success allows conditional knockout and the removal of selectable markers without prior parasite modification, which also facilitate the transferring of DiCRE recombinase to different T. cruzi strains.

3] Analysis of mammalian cis-regulatory DNA elements by homologous recombination

The use of homologous recombination to modify and thereby functionally analyze cis-regulatory DNA elements in mammalian cells has become an important approach in mammalian gene expression research. We have emphasized the necessity of designing a system that allows the removal of selectable markers used in targeting and facilitates the further modification of the region under study. To perform these tasks, we presently favor making an initial HR-mediated replacement of the entire element under study with an active positive selectable marker in combination with either an inactive second positive selectable marker or an active negative selectable marker. The plug and socket system, in which an inactive selectable marker is complemented by HR, is the most dependable and well-characterized option for making secondary modifications. However, the double-replacement system has certain advantages, and the recently developed RMCE approach, which allows replacement of a negative selectable marker by site-specific recombinase-mediated insertion without using a positive selectable marker, will likely prove very valuable in future experiments. Each of the systems, or combinations thereof, should be considered in light of the specifics of any given experiment to select the most appropriate option. Although the emphasis of this article has been the analysis of cis-acting regulatory elements involved in transcription, these same approaches can be used to analyze other regulatory elements (e.g., origins of replication) and to make multiple subtle mutations in polypeptides.