Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail.

Zygotes at the 1-cell stage have been genetically modified by microinjecting the CRISPR/Cas9 components for the generation of targeted gene knockout in mammals. In the avian species, genetic modification of the zygote is difficult because its unique reproductive system limits the accessibility of the zygote at the 1-cell stage. To date, only a few CRISPR/Cas9-mediated gene knockouts have been reported using the chicken as a model among avian species, which requires 3 major processes: isolation and culture of primordial germ cells (PGCs), modification of the genome of PGCs in vitro, and injection of the PGCs into the extraembryonic blood vessel at the early embryonic stages when endogenous PGCs migrate through circulation to the genital ridge. In the present study, the adenoviral CRISPR/Cas9 vector was directly injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin (MLPH) gene, which is involved in melanosome transportation and feather pigmentation. MLPH homozygous mutant quail exhibited gray plumage, whereas MLPH heterozygous mutants and wild-type quail exhibited dark brown plumage. In addition, the adenoviral vector was not integrated into the genome of knockout quail, and no mutations were detected in potential off-target regions. This method of generating genome-edited poultry is expected to accelerate avian research and has potential applications for developing superior genetic lines for poultry production in the industry.

Molecular cloning, structural analysis and tissue expression of protein phosphatase 3 catalytic subunit alpha isoform (PPP3CA) gene in Tianfu goat muscle.

Calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase, plays a critical role in controlling skeletal muscle fiber type. However, little information is available concerning the expression of calcineurin in goat. Therefore, protein phosphatase 3 catalytic subunit alpha isoform (PPP3CA) gene, also called calcineurin Aα, was cloned and its expression characterized in Tianfu goat muscle. Real time quantitative polymerase chain reaction (RT-qPCR) analyses revealed that Tianfu goat PPP3CA was detected in cardiac muscle, biceps femoris muscle, abdominal muscle, longissimus dors muscle, and soleus muscle. High expression levels were found in biceps femoris muscle, longissimus muscle and abdominal muscle (p < 0.01), and low expression levels were seen in cardiac muscle and soleus muscle (p > 0.05). In addition, the spatial-temporal mRNA expression levels showed different variation trends in different muscles with the age of the goats. Western blotting further revealed that PPP3CA protein was expressed in the above-mentioned tissues, with the highest level in biceps femoris muscle, and the lowest level in soleus muscle. In this study, we isolated the full-length coding sequence of Tianfu goat PPP3CA gene, analyzed its structure, and investigated its expression in different muscle tissues from different age stages. These results provide a foundation for understanding the function of the PPP3CA gene in goats.

Molecular cloning, sequence identification and expression analysis of novel caprine MYLPF gene.

Skeletal muscle genes are important potentially functional candidate genes for livestock production and meat quality. Myosin regulatory light chain (MLC) regulates myofilament activation via phosphorylation by Ca(2+) dependent myosin light chain kinase. The cDNA of the myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF) gene from the longissimus dorsi of Tianfu goat was cloned and sequenced. The results showed that MYLPF full-length coding sequence consists of 513 bp and encodes 170 amino acids with a molecular mass of 19.0 kD. Two EF-hand superfamily domain of MYLPF gene conserved between caprine and other animals. The deduced amino acid sequence of MYLPF shared significant identity with the MYLPF from other mammals. A phylogenetic tree analysis revealed that the caprine MYLPF protein has a close genetic relationship and evolutional distance with MYLPF in other mammals. Analysis by RT-PCR showed that the MYLPF mRNA was detected in heart, liver, spleen, lung, kidney, gastrocnemius, abdominal muscle and longissimus dorsi. In particular, high expression levels of MYLPF mRNA were detected in the longissimus dorsi, gastrocnemius and abdominal muscle, and low level of expressions were observed in liver, spleen, lung and kidney. In addition, the temporal expression analysis further showed MYLPF expression decreased gradually with age in the skeletal muscle. This may be important as muscle growth occurs mainly in young age in goats. Western blotting results detected the MYLPF protein in four of the tissues in which MYLPF was shown to be expressed; the four exceptions were liver, spleen, lung and kidney.

Molecular cloning, sequence analysis and tissue-specific expression of Akirin2 gene in Tianfu goat.

The Akirin2 gene is a nuclear factor and is considered as a potential functional candidate gene for meat quality. To better understand the structures and functions of Akirin2 gene, the cDNA of the Tianfu goat Akirin2 gene was cloned. Sequence analysis showed that the Tianfu goat Akirin2 cDNA full coding sequence (CDS) contains 579bp nucleotides that encode 192 amino acids. A phylogenic tree of the Akirin2 protein sequence from the Tianfu goat and other species revealed that the Tianfu goat Akirin2 was closely related with cattle and sheep Akirin2. RT-qPCR analysis showed that Akirin2 was expressed in the myocardium, liver, spleen, lung, kidney, leg muscle, abdominal muscle and the longissimus dorsi muscle. Especially, high expression levels of Akirin2 were detected in the spleen, lung, and kidney whereas lower expression levels were seen in the liver, myocardium, leg muscle, abdominal muscle and longissimus dorsi muscle. Temporal mRNA expression showed that Akirin2 expression levels in the longissimus dorsi muscle, first increased then decreased from day 1 to month 12. Western blotting results showed that the Akirin2 protein was only detected in the lung and three skeletal muscle tissues.

Molecular cloning and characterization of different expression of MYOZ2 and MYOZ3 in Tianfu goat.

The myozenin family of proteins binds calcineurin, which is involved in myocyte differentiation of skeletal muscle. Moreover, gene expression of myozenin is closely related to meat quality. To further understand the functions and effects of myozenin2 (MYOZ2) and myozenin3 (MYOZ3) genes in goat, we cloned them from Tianfu goat longissimus dorsi muscle. Sequence analyses revealed that full-length coding sequence of MYOZ2 consisted of 795 bp and encoded 264 amino acids, and full-length coding sequence of MYOZ3 consisted of 735 bp and encoded 244 amino acids. RT-qPCR analyses revealed that mRNA expressions of MYOZ2 and MYOZ3 were detected in heart, liver, spleen, lung, kidney, leg muscle, abdominal muscle, and longissimus dorsi muscle. Particularly high expression levels of MYOZ2 were seen in abdominal muscle and heart (P<0.01), low expression levels were seen in leg muscle (P<0.01), longissimus dorsi muscle (P>0.05) and very little expression were detected in liver, spleen, lung and kidney (P>0.05). In addition, high expression levels of MYOZ3 were seen in abdominal muscle, leg muscle, lungs and kidney (P<0.01), low expression levels were found in longissimus dorsi muscle and spleen (P<0.01) and very little expression were detected in heart and liver (P>0.05). Temporal mRNA expression results showed that MYOZ2 and MYOZ3 gene expression varied across four muscle tissues with different ages of the goats. Western blotting further revealed that MYOZ2 and MYOZ3 proteins were only expressed in goat muscle, with notable temporal expression differences in specialized muscle tissues from five development age stages. This work provides the first evidence that MYOZ2 and MYOZ3 genes are expressed abundantly in Tianfu goat muscle tissues from different development age stages, and lay a foundation for understanding the functions of MYOZ2 and MYOZ3 genes in muscle fiber differentiation.