Uses and kits of CFAP61 in diagnosing or predicting birth defects
A kit and reagent technology, applied in the field of disease and diagnosis, which can solve problems such as elusive etiology
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Embodiment 1
[0058] Embodiment 1 experimental method and reagent
[0059] Genotyping
[0060] Genotyping was performed by PCR amplification of genomic DNA extracted from mouse tails.
[0061] The PCR of the Cfap61 mutant allele produces a 549bp fragment, and the primers are as follows:
[0062] Forward: 5-CCTCGGTGGTTTCTCCTATAAGTTC-3,
[0063] Reverse: 5-AGGGTAGTGTTGTGCCTGTGA-3.
[0064] PCR of the wild-type allele of Cfap61 produces a 406bp fragment, and the primers are as follows:
[0065] Forward: 5-GCAGGACTGTGTTCAAGGCGGTG-3,
[0066] Reverse: 5-AGGTAGTGATTTGTGCCTGTGAC-3.
[0067] real-time RT-PCR
[0068] Total RNA was isolated from various tissues of wild-type or knockout adult mice. To analyze the expression levels of Cfap61 mRNA in various tissues, equal amounts of cDNA were synthesized using PrimeScript RT Kit with gDNA Eraser (Takara). qPCR was performed using TB Green Premix Ex Taq (Takara) and specific forward and reverse primers: Cfap61 primer pair F 5'-CTGTTTGGGAGGCTCAA...
Embodiment 2
[0090] Example 2 Quantitative RT-PCR detection of expression of mouse testis-specific gene Cfap61
[0091] To determine the expression pattern in mice, RT-PCR of Cfap61 was performed in multiple tissues of adult mice ( figure 1 a). This analysis revealed that Cfap61 is abundantly expressed in the testis but not in other organs. To verify the transient expression of Cfap61 associated with spermatogenesis, RT-PCR was performed using postpartum testes ( figure 1 b). Cfap61 was shown to be expressed from postnatal day 25, which corresponds to the period of condensation and sperm flagella formation during spermatogenesis.
Embodiment 3
[0092] Example 3 Preparation of Cfap61-deficient mouse model
[0093] Mouse Cfap61 gene (Transcript: ENSMUSG00000037143), 71.28kb in length, is located on chromosome 2. Thirty-one exons have been identified, with exon 2 being the ATG start codon and exon 17 being the TGA stop codon. To study the in vivo function of CFAP61, Cfap61 knockout mice were generated using the CRISPR-Cas9 system targeting the Cfap61 locus with deletion of exon 2 to exon 5 ( figure 1 c), Generation of a Cfap61-deficient mouse model.
[0094] Specifically, the CRISPR / Cas9-mediated genome editing system of Cyagen Biosciences was used. Exon 3 and exon 5 were selected as target sites. The gRNA and Cas9 mRNA were co-injected into fertilized eggs of C57BL / 6 mice to generate a targeted strain with a 4227 bp base deletion, ATATCCCGAGTACCACCTCAGTCTTAGCAAAGGACAGTTG-del4227bp-TGGAATGGGGACCACAATTTGTTCTGAAATCCTAAGCTA. The founder animal was identified by DNA sequence analysis. Mice heterozygous for the target c...
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