Abstract
The mouse artificial chromosome (MAC) has several advantages as a gene delivery vector, including stable episomal maintenance of the exogenous genetic material and the ability to carry large and/or multiple gene inserts including their regulatory elements. Previously, a MAC containing multi-integration site (MI-MAC) was generated to facilitate transfer of multiple genes into desired cells. To generate transchromosomic (Tc) mice containing a MI-MAC with genes of interest, the desired genes were inserted into MI-MAC in CHO cells, and then the MI-MAC was transferred to mouse embryonic stem (mES) cells via microcell-mediated chromosome transfer (MMCT). However, the efficiency of MMCT from CHO to mES cells is very low (<10−6). In this study, we constructed mES cell lines containing a MI-MAC vector to directly insert a gene of interest into the MI-MAC in mES cells via a simple transfection method for Tc mouse generation. The recombination rate of the GFP gene at each attachment site (FRT, PhiC31attP, R4attP, TP901-1attP and Bxb1attP) on MI-MAC was greater than 50 % in MI-MAC mES cells. Chimeric mice with high coat colour chimerism were generated from the MI-MAC mES cell lines and germline transmission from the chimera was observed. As an example for the generation of Tc mice with a desired gene by the MI-MAC mES approach, a Tc mouse strain ubiquitously expressing Emerald luciferase was efficiently established. Thus, the findings suggest that this new Tc strategy employing mES cells and a MI-MAC vector is efficient and useful for animal transgenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-015-9884-6) contains supplementary material, which is available to authorized users.
Highlights
The mouse artificial chromosome (MAC) has several advantages as a gene delivery vector, including stable episomal maintenance of the exogenous genetic material and the ability to carry large and/ or multiple gene inserts including their regulatory elements
Twenty-five clones were obtained, and these mouse embryonic stem (mES) cell clones were checked by genomic PCR, and FISH analysis for retention of the intact MI-MAC (Fig. 1c)
The germline transmission (GT) rate of female F1 MI-MAC Tc mice derived from MI-MAC mES cell line 4 and 5 was 45.3 and 57.1 %, respectively
Summary
The mouse artificial chromosome (MAC) has several advantages as a gene delivery vector, including stable episomal maintenance of the exogenous genetic material and the ability to carry large and/ or multiple gene inserts including their regulatory elements. To generate transchromosomic (Tc) mice containing a MI-MAC with genes of interest, the desired genes were inserted into MI-MAC in CHO cells, and the MI-MAC was transferred to mouse embryonic stem (mES) cells via microcell-mediated chromosome transfer (MMCT). As an example for the generation of Tc mice with a desired gene by the MI-MAC mES approach, a Tc mouse strain ubiquitously expressing Emerald luciferase was efficiently established. Expression of the gene of interest is susceptible to positional effects To overcome this problem, the knock in (KI) of one copy of a desired gene at a defined locus such as the Rosa or Hprt gene region by homologous recombination in mouse embryonic stem (mES) cells has been used to generate Tg mice stably expressing the gene (Soriano 1999; Yang et al 2009). In the KI approach, Mb-sized and multiple different genes cannot be transferred to a single locus
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
