Identification of the Transgene Integration Site and Host Genome Changes in MRP8-Cre/ires-EGFP Transgenic Mice by Targeted Locus Amplification

Abstract

The MRP8-Cre-ires/EGFP transgenic mouse (Mrp8cre^Tg, on C57BL/6J genetic background) is popular in immunological and hematological research for specifically expressing Cre recombinase and an EGFP reporter in neutrophils. It is often crossed with other transgenic lines carrying loxP-flanked genes to achieve restricted gene knockout in neutrophils. However, due to the way in which the line was created, basic knowledge about the MRP8-Cre-ires/EGFP transgene in the host genome, such as its integration site(s) and flanking sequences, remains largely unknown, hampering robust experimental design and data interpretation. Here we used a recently developed technique, targeted locus amplification (TLA) sequencing, to fill these knowledge gaps. We found that the MRP8-Cre-ires/EGFP transgene was integrated into chromosome 5 (5qG2) of the host mouse genome. This integration led to a 44 kb deletion of the host genomic sequence, resulting in complete deletion of Serpine1 and partial deletion of Ap1s1. Having determined the flanking sequences of the transgene, we designed a new genotyping protocol that can distinguish homozygous, heterozygous, and wildtype Mrp8cre^Tg mice. To our surprise, crossing heterozygous mice produced no homozygous Mrp8cre^Tg mice, most likely due to prenatal lethality resulting from disrupted Ap1s1 gene expression.

Keywords: MRP8-Cre transgenic mouse; Cre-loxP system; TLA sequencing; homozygous lethality; neutrophil.

Figure 1

The integration site of the hMRP8-Cre/ires-EGFP transgene on mouse chromosome 5 (Chr 5) and the DNA sequences of the connecting regions between the mouse genome and human MRP8. The hMRP8-Cre/ires-EGFP transgene was integrated into the mouse genome within the intronic region of Ap1s1 between exons 2 and 3 on chromosome 5, resulting in a 44 kb (Δ44 kb) deletion of the mouse genome containing the full sequence of Serpine1 and a partial sequence of Ap1s1. (A) The schematic shows wild-type C57BL/6 mouse Chr 5 (the blue line) with intact Ap1s1 and Serpine1 and the altered Chr 5 in Mrp8cre^Tg mice with the inserted hMRP8-Cre/ires-EGFP transgene. The dotted blue line indicates a 44 kb deletion of the mouse genome on Chr 5 (cartoons not drawn to scale). Green boxes indicate exons. (B) DNA sequences of the connecting regions between the mouse genome and the integrated hMRP8-Cre/ires-EGFP transgene. Letters in blue indicate the mouse genomic sequence, while letters in black indicate the hMRP8-Cre/ires-EGFP transgenic sequence.

Figure 2

A new genotyping protocol distinguishes the different genotypes of Mrp8cre^Tg mice. (A) The primer set designed for Mrp8cre^Tg mouse genotyping based on TLA sequencing results (upper panel). A representative DNA gel genotyping two litters of Mrp8cre^Tg mice bred by mating male heterozygotes with female heterozygotes. Two DNA bands of 574 bp and 230 bp are observed for heterozygous pups, while only one 574 bp band is observed for wildtype pups (lower panel). (B) Amongst 68 Mrp8cre^Tg pups bred from heterozygous parents, 44 heterozygous versus 24 wildtypes were detected and no homozygous pups were found.