Bacterial artificial chromosome (BAC) for the TTC9A gene was obtained from BACPAC resources centre (https://bacpacresources.org/). After which Gene Bridges, Quick and Easy BAC modification protocol (https://www.genebridges.com/home) was used to modify the TTC9A gene. Briefly, the TTC9A gene comprises of 3 exons. We deleted exon 1 (433 bp) which contained 70% of the coding region and replaced it with a neomycin cassette. This neomycin cassette was later used as a positive selection marker for identification of transgenic sequence in the mouse embryonic stem. The modified BAC was subcloned into minimal vector which contained the ampicillin resistance marker for bacterial selection. After the transgene was subcloned, it was screened and sequenced. The construct known as “TTC9A target construct” was linearized and electroporated into mES cells passage 11 (R1/E (ATCC SCRC-1036). The mES R1 cells were maintained and screened for positive selection using G418 (Geneticin). Resistance to G418 is conferred by the Neomycin resistance gene (neo), which is incorporated in the TTC9A target construct. We used digoxygenin DNA labeling kit for southern blot and PCR techniques, numerous mES cells were screened to confirm successful homologous recombination and integration of the TTC9A target construct in the DNA of the mES cells. Finally the “targeted” mES cells which contained the TTC9A modified locus was identified. The homologous recombination efficiency was 10% for the TTC9A locus. Before microinjection these modified mES cells were karyotyped and was at passage 13-15.

R1/E

TTC9A Knockout mice generation strategy: C57BL6 male and C57BL6 female mice were mated. Blastocysts of the C57BL6 (Black) was microinjected with the TTC9A targeted ES cells of R1 (agouti) background. Transgenic ES cells integrate into the embryo. CD-1 pseudo pregnant mother uterus was used to re-implant the modified embryos. Peudopregnancy in CD-1 female mice was attained using vasectomised CD-1 male mice. Chimeric progeny mice (Black and agouti chimeric coat colour) were obtained. Chimeric mice was crossed with normal C57BL6 mice. To confirm germ line transmission pups were screened for heterozygous (agouti) genotype. These were verified by both polymerase change reaction (PCR) and southern blotting (SB). Lastly, the heterozygous mice were crossed to obtain homozygous wild type (TTC9A+/+), homozygous transgenic (TTC9A-/-) and heterozygous transgenic (TTC9A+/-). Chimera status: Four mouse embryonic stem (mES) clones containing the TTC9A transgene TTC9A_A1, TTC9A_F9, TTC9A_c103, and TTC9A_c128 were karyotyped and then were used for microinjection. From the four clones TTC9A_c128 gave two chimeras. Chimera 1 (TTC9A_c128_M1) was microinjected on 18/12/08 with 1036-R1 TTC9A transgene mES and was 100 % chimeric, with DOB: 1/6/2009. It later gave birth (DOB: 15/3/2009) to 5 agouti pups (m1.1a, m1.3a, m1.5a, f1.0a, f1.2a) of which m1.3a, m1.5a were confirmed to be heterozygous using PCR and southern blot (Figure 1). Chimera 2 (TTC9A_c128_M2) was microinjected on 18/12/08 with and gave a 97 % chimeric mice, with the DOB: 1/6/2009. It later gave birth (DOB: 4/7/2009) to 9 agouti pups (m2.1a, m2.3a, m2.5a, m2.7a, m2.9a, f2.0a, f2.2a, f2.4a, f2.6a) and of which m2.1a, m2.3a, m2.5a, m2.7a, m2.9a, f2.0a, f2.4a were heterozygous. Note this was an ex runt and it died on the 4/4/09. Het mice obtained from TTC9A_c128_M1 was taken forward for getting homozygous mouse lines. These heterozygous male mice were crossed with C57BL6 mice for 5-7 generations later. The chimera mice were bred to C57BL6 mouse line. Backcrossing of chimera to C57BL6. On 26/5/2009 m1.3a, m1.5a, m1.11a, m2.1a, m2.3a heterozygous males, which were obtained from chimeric mice 1 and 2, were crossed with C57BL6 mice. Only m1.5a and m1.11a gave birth to 12 and 4 pups respectively. Fertility check crosses between TTC9A heterozygous mice. We also performed a heterozygous crossing to confirm the fertility of these TTC9A heterozygous mouse lines. Heterozygous TTC9A male (m1.3a, m1.5a, m1.11a, m2.1a, m2.3a) mice were crossed with heterozygous TTC9A females (f1.4a, f2.0a, f2.4a , f2.2.16b). These gave birth to 77 pups with the male: female ratio of 32:45 and wild: het: homo ratio of 30:29:18. With average litter size of 8 pups. TTC9A heterozygous and homozygous female and TTC9A heterozygous male fertility check. To further confirm the fertility status of the TTC9a homozygous females (f5.2.0, f2.2.12a), they were crossed with het males (m1.3a and m1.5a). The first pair (m1.3a X f5.2.0) gave birth on 18/8/2009, while the second pair (m1.5a X f2.2.12a) gave birth on 17/8/2009. These two pairs gave birth to 14 pups (8+6), male: female ratio of 7:7, heterozygous to homozygous (Ht:Ho) ratio of 9:5. TTC9A homozygous male and female fertility check. Three TTC9a homozygous males (m2.2.23a, m1.2.9a and m3.2.5a) were crossed with TTC9a homozygous females (f5.2.0, f2.2.12a, f2.2.26a and f1.2.2a). In total 23 pups were born. The male to female ratio was 10:13. All were genotyped by pcr and southern blot to confirm homozygosity. All animal experiments were conducted under the IACUC guidelines (Institutional animal care and use committee). The animals were maintained under standard housing conditions in line with the guidelines of the FELASA (Federation of Laboratory Animal Science Associations).

Wild-type littermates

The MMRRC Centers have developed a genetic QC pipeline using MiniMUGA array genotyping to provide additional information on strain backgrounds for MMRRC congenic and inbred strains. For more information on when data may be available, or to request genotyping for a strain of interest, please contact csmmrrc@jax.org. Older strains may not have this information.

Chun Ling Valerie Lin, Ph.D., Nanyang Technological University.