A novel genotyping method to determine copy number in a mouse line commonly used for inducible transgene expression in brain and spinal cord

Ethical statement

All animal work was approved following local ethical review by the University of Manchester Animal Welfare and Ethical Review Board and performed under Home Office project license 70/8903 and in accordance with the Home Office (Animals) Scientific Procedures Act (1986). All efforts were made to ameliorate harm to animals; mice were housed and maintained according to standard practices in the University of Manchester Biological Services Facility (detailed below) and no licenced procedures were performed.

Maintenance of transgenic mice

B6;C3-Tg(NEFH-tTA)8Vle/J mice (JAX stock #025397) were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and bred with C57BL6/J mice to produce a colony of hemizygous NEFH-tTA animals (13 transgenic mice were crossed with 12 wild-type in total, as this number was sufficient to create a new colony of backcrossed mice). All animal work was carried out in the University of Manchester Biological Services Facility. Mice had free access to food and water and were housed under light-humidity- and temperature-controlled conditions: ambient temperatures of 21°C (±2°C), humidity of 40–50%, 12 h light cycle, ad libitum access to water and standard rodent chow. Animals were housed up to five per cage with environmental enrichment. For breeding, mice were housed as either breeding pairs or trios of two female and one male, and pups housed with parents until weaning (approximately four weeks post-birth).

At weaning, animals were restrained by scruffing and ear biopsies were taken using a standard ear puncher and genotyped as described¹. Briefly, tissue was disrupted in proteinase K for one hour at 57°C; reactions containing 3μL of diluted DNA, 10μL PrimeSTAR HS Premix (Takara, Clontech #R040A) and 0.5μM each of forward and reverse transgene primers and internal positive control primers (Primers 1–4, Table 1) in a total volume of 20μL were amplified in a thermal cycler (ThermoFisher SimpliAmp^TM A24812; Table 2). PCR products were visualised on a 2% agarose gel with HyperLadder IV (Bioline) to determine product size.

Identification of the transgene insertion point

A combination of targeted locus amplification (TLA) and next-generation sequencing was used to identify the insertion point of the NEFH-tTA transgene (the FASTA of the transgene sequence can be viewed in the Underlying data). This was performed by Cergentis B.V (Utrecht, The Netherlands). One mouse spleen was dissected from a six-week old NEFH-tTA^+/- mouse (culled by Schedule 1 CO₂ inhalation) and cells isolated for TLA sample prep. Spleen tissue was disrupted by gently pushing through a 40µm mesh and cells collected in PBS with 10% foetal calf serum (Gibco #10500064). Cells were pelleted by centrifugation at 250g for ten minutes at room temperature, resuspended in lysis buffer (0.15M NH₄CL, 0.01M KHCO₃, 0.0002M EDTA) and incubated at room temperature for five minutes before additional centrifugation at 250g for ten minutes. Cells were resuspended in PBS with 10% foetal calf serum (Gibco #10500064), stored at -80°C and shipped to Cergentis on dry ice.

Two primer sets (Table 3) were used in individual TLA amplifications. PCR products were purified and library prepped using the Illumina NexteraXT protocol and sequenced on an Illumina sequencer. Reads were mapped using BWA-SW, which is a Smith-Waterman alignment tool. This allows partial mapping, which is optimally suited for identifying break-spanning reads. The mouse genome version mm10 (GenBank assembly accession: GCA_000001635.2) was used for mapping.

Genotyping of offspring from hemizygous intercross matings

Hemizygous mice were bred together (total 25 animals; the number of breeding animals was chosen based on expected number of homozygous offspring and numbers required to set up a large colony to breed with a different transgenic line³) and ear biopsies taken from all offspring at weaning (n=50) as described above. Primers were designed using Primer3 (web version 4.1.0) to produce a product spanning the insertion site on chromosome 12 (Primers 5-6, Table 1) and PCR reactions were set up as described under Maintenance of transgenic mice. REDExtract-N-Amp™ 2X PCR Reaction Mix (Sigma-Aldrich, #R4775) was used instead of PrimeStar.

Statistical analysis

Outcome vs Expected for genotype was performed using GraphPad Prism version 8.00 for Mac (GraphPad Software, La Jolla California USA, www.graphpad.com).

The NEFH-tTA transgene is located on chromosome 12

Using TLA highest coverage is observed on the sequences directly surrounding the location of the primer set. Here, high coverage is observed on chromosome 12, outlined in red in Figure 1A, indicating the transgene (TG) has integrated in chromosome 12⁴.

Figure 1.

(A) Targeted locus amplification (TLA) sequence coverage across the mouse genome using primer set 1. The chromosomes are indicated on the y-axis, the chromosomal position on the x-axis. Similar results were obtained with primer set 2 (see Underlying data⁴). (B) TLA sequence coverage across mouse chromosome 12 with primer pair 1. The arrow points towards the integration site. (C) TLA sequence coverage across the TG with primer pair 1 (top) and 2 (bottom).

From locus-wide coverage (Figure 1B) we were able to identify the junction sites between the TG and the mouse genomic sequence, and it was concluded that the TG has integrated in mouse chr12:6917896-chr12:6917912. Reads marking the TG integration are identified in Table 4. The 11 bases between chr12:6917896-chr12:6917912 have been deleted following the integration event. According to the reference sequence (mm10) there are no genes annotated in this region. Complete coverage is observed across the whole TG sequence from TG:68-18543 (Figure 1C). At the edges of the coverage, fusion reads have been found, connecting these ends, but the TG: 1 -68 and TG 18544 – end regions are not integrated into the mouse genome (Table 4). These regions contained sequences for bacterial DNA replication and therefore are not required for transgenic expression in the mouse.

Next to the head-to-tail fusion as reported above, four other TG-TG fusions were found within the TG sequence (Table 4). While an exact copy number cannot be determined using TLA, an estimation can be made based on the number of integration sites, number of fusion reads and the ratio of coverage on the TG and genome integration site. Here, one integration site was found and a total of five TG-TG fusions were found. The coverage at the TG was significantly higher compared to the genome. These facts together suggest that >5 copies of the TG may have integrated.

Design of a PCR assay to distinguish between hemizygous and homozygous transgenic mice

We designed primers to amplify a wild-type PCR product of 488bp from chromosome 12, which would be disrupted by insertion of the transgenic allele (Figure 2A). A multiplex PCR reaction including the original primers to the transgene¹ (Table 1) was carried out on samples from a hemizygous intercross and proved able to distinguish between wild-type (488bp band only), hemizygous (both bands) and homozygous (150bp band only) (Figure 2B)⁴.

Figure 2. Genotyping strategy for homozygous mice.

(A) Primer design to produce a wild-type product that is disrupted in the presence of the transgenic allele. (B) Samples visualised on a 2% agarose gel showing wild-type (top band), homozygous (bottom band) and hemizygous (both bands) animals.

Homozygous NEFH-tTA mice are observed at the same rate as expected

After genotyping 50 offspring of hemizygous intercrosses we observed no significant difference in the number of Nefh-tTA^+/+ mice compared to the expected ratio (Table 5, chi-square=2.64, p<0.2671⁴), confirming our novel genotyping assay performs as expected. Homozygous mice grew to adulthood and did not exhibit any overt phenotype.

Underlying data

Figshare: “A novel genotyping method to determine zygosity in a mouse line commonly used for inducible transgene expression in brain and spinal cord – Raw data/analysis” DOI: https://doi.org/10.6084/m9.figshare.12982220.v1⁴.

This project contains the following underlying data:

Figshare: FASTA reference file for the NEFH-tTA transgene, https://doi.org/10.6084/m9.figshare.16847170.v1⁵

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).