Strain Name:
B6.129-Ttc9tm1Vcl/Mmjax
Stock Number:
043521-JAX
Citation ID:
RRID:MMRRC_043521-JAX
Other Names:
TTC9AKO,

Gene Information

Ttc9tm1Vcl
Name: tetratricopeptide repeat domain 9; targeted mutation 1, Valerie Lin
Synonyms: Ttc9a-
Type: Allele
Species: Mus musculus (mouse)
Chromosome: 12
Alteration at locus: Knockout
Ttc9
Name: tetratricopeptide repeat domain 9
Synonyms: 1700029M07Rik
Type: Gene
Species: Mus musculus (mouse)
Chromosome: 12
Alteration at locus: Knockout
NCBI: 69480
VEGA: 12
Homologene: 52649
Genetic Alterations
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.
Genotype Determination
ES Cell Line
R1/E
Phenotype

Homozygous: There is no obvious phenotype in the TTC9A KO Homozygous mice

Hetero/Hemizygous: There is no obvious phenotype in the TTC9A KO Heterozygous/Hemizygous mice

Cre-excised Phenotype: Undetermined 

Mammalian Phenotype Terms
Allelic Composition: (Genetic Background: )

MeSH Terms
  • Animals
  • Estrogen Receptor alpha/genetics
  • Estrogen Receptor alpha/metabolism
  • Female
  • Humans
  • MCF-7 Cells
  • Mammary Glands, Human/metabolism
  • Mice
  • Mice, Knockout
  • Microfilament Proteins/genetics
  • Microfilament Proteins/metabolism
  • Nerve Tissue Proteins/genetics
  • Nerve Tissue Proteins/metabolism
  • Protein Binding
  • Signal Transduction
  • Tacrolimus Binding Proteins/metabolism
Strain Development
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).
Suggested Control Mice
Wild-type littermates
  • Cancer
  • Cell Biology
  • Developmental Biology
  • Endocrine Deficiency
  • Immunology and Inflammation
  • Reproduction
Donor
Chun Ling Valerie Lin, Ph.D., Nanyang Technological University.
Primary Reference

Shrestha S, Sun Y, Lufkin T, Kraus P, Or Y, Garcia YA, Guy N, Ramos P, Cox MB, Tay F, Lin VC. Tetratricopeptide repeat domain 9A negatively regulates estrogen receptor alpha activity. Int J Biol Sci. 2015 Feb 27;11(4):434-47. (Medline PMID: 25798063)

Colony and Husbandry Information

Mice recovered from a cryo-archive will have health surveillance performed on recipient females. Health reports will be provided prior to shipment. If you require additional health status information, please email csmmrrc@jax.org .
Coat Color
Black
MMRRC Breeding System
Backcross
Generation
5 - 7 backcrosses were performed on the heterozygous mice from TTC9A_c128_M1 background.
Overall Breeding Performance
Good
Viability and Fertility: Female Male Comments
Homozygotes are viable: Yes Yes
Homozygotes are fertile: Yes Yes
Heterozygotes are fertile: Yes Yes
Age Reproductive Decline: Undetermined Undetermined
Average litter size
4-6
Recommended wean age
3-4 weeks

Order Request Information

Limited quantities of breeder mice (recovered litter) are available from a cryoarchive; recovered litter usually available to ship in 3 to 4 months.

Cryopreserved material may be available upon request, please inquire to csmmrrc@jax.org for more information.

Distribution of this strain requires submission of the MMRRC Conditions of Use (COU). A link to the COU web form will be provided via email after an order has been placed; the form should be completed then or the email forwarded to your institutional official for completion.

The donor or their institution limits the distribution to non-profit institutions only.

Additional charges may apply for any special requests. Shipping costs are in addition to the basic distribution/resuscitation fees. Information on shipping costs and any additional charges will be provided by the supplying MMRRC facility.

Click button to Request this one strain. (Use the MMRRC Catalog Search to request more than one strain.)
MMRRC Item # Description Distribution Fee / Unit (US $) Units Notes
043521-JAX-SPERM Cryo-preserved spermatozoa $437.00 / Non-Profit Aliquot Approximate quantity3
043521-JAX-RESUS Litter recovered from cryo-archive $2,022.00 / Non-Profit Litter Recovered litter4; additional fees for any special requests.
Cryopreserved material may be available upon request, please inquire to csmmrrc@jax.org for more information.

1 The distribution fee covers the expense of rederiving mice from a live mouse; you will receive the resulting litter. The litter will contain at minimum one mutant carrier; the actual number of animals and the gender and genotype ratios will vary. (Typically, multiple breeder pairs can be established from the recovered litter.) Prior to shipment, the MMRRC will provide information about the animals recovered. If you anticipate or find that you need to request specific genotypes, genders or quantities of mice in excess of what is likely from a resuscitated litter, you may discuss available options and pricing with the supplying MMRRC facility.

2 An aliquot contains a sufficient number of embryos (in one or more vials or straws and based on the transfer success rate of the MMRRC facility) to transfer into one to three recipients. The MMRRC makes no guarantee concerning embryo transfer success experienced in the recipient investigator's laboratory. Neither gender nor genotype ratios are guaranteed.

3 An aliquot is one straw or vial with sufficient sperm to recover at least one litter of mice, as per provided protocols, when performed at the MMRRC facility. The MMRRC makes no guarantee concerning the success of these procedures when performed outside the MMRRC facilities.

4 The distribution fee covers the expense of resuscitating mice from the cryo-archive; you will receive the resulting litter. The litter will contain at minimum one mutant carrier; the actual number of animals and the gender and genotype ratios will vary. (Typically, multiple breeder pairs can be established from the recovered litter.) Prior to shipment, the MMRRC will provide information about the animals recovered. If you anticipate or find that you need to request specific genotypes, genders or quantities of mice in excess of what is likely from a resuscitated litter, you may discuss available options and pricing with the supplying MMRRC facility.

To request material from the MMRRC: Please fill out our on-line request form (accessible from the catalog search results page, or click the Request this Strain button in the fees section). If you have questions or need assistance completing this form, you may call Customer Service at (800) 910-2291 (in USA or Canada) or (530) 757-5710 (international calls). Before you call, please have with you: the MMRRC item number, quantity needed, Bill-to and Ship-to contact information.