Generation of transgenic ducks by crispr/CAS9-mediated gene inser-tion combined with the sperm-mediated gene transfer (SMGT)

Abstract

To edit the duck genome by HDR-directed integration of the EGFP gene into the duck host genome in combination with SMGT using CRISPR/Cas9. Methods. HDR-mediated gene of green fluorescent protein (EGFP) was crried out by the combined action of four plasmids. The pX330 contained the Cas9 gene. Two plasmids contained sgRNA genes: pBR322-sgRNA1 and pBR322-sgRNA2. The pBR322-HDR-EGFP plasmid was constructed to contain the DNA vector with left homologous sequence part(LHP), the EGFP gene coding sequences and the right homologous sequence part(RHP). The DNA sequence data for designing the HDR-EGFPinsert and sgRNA 1 and sgRNA 2 were taken from the genome DNA sequence of Anas platyrhynchos Spindlin 1 (SPIN1) gene. Twenty four ducks (13 males and 11 females) of the Shaoxing breed were used for this experiment. The sperm transfection was performed using Lipofectamine 2000. Results. Thirty one ducks were obtained, 19 of which carried the EGFP gene. F2 analysis revealed that 16 ducks (F1) (14 females and 2 males) transmitted the transgene DNA to their offsprings. Thus 27.6 % (56/203) of F2 descendants were positive for the transgene DNA construct. Conclusions. Exogenous DNA was successfully inserted into the duck genome.

Efficacy of blastodermal cells and CRISPR/CAS9 method in the creation of transgenic duck (Anas Platyrhynchos)

“…1. Scheme of the experiment on the creation of transgenic chimeras more detail in our previous work, where we used sperm to create transgenic ducks [17,47].…”

“…Three methods are mostly used to obtain transgenic birds: 1) transfection of the DNA vector with sperm [17,18]; 2) DNA injections into the embryonic cavity of a newly laid egg [19][20][21]; 3) introduction of donor cells [22][23][24]. Certain complications have been established in the implementation of the transgene with the introduction of DNA vector using the method of transfection with sperm [17].…”

Genome editing and its applications in genetic improvement in aquaculture

“…electroporation 69 ) to allow for more efficient delivery of gRNA and Cas into large numbers of eggs for breeding purposes. Other effective ways to deliver gRNA-Cas9 into fertilised eggs must be developed and tried, such as sperm-mediated transfer, 109 microinjection of unfertilized ova 110 and editing primordial germ cells (Figure 4), which was used to edit genes in chickens 111 and zebrafish. 112 Germ cell transplantation from fish with a desired edit, into multiple sterile surrogates enables the enrichment of the edited alleles of interest (Figure 4), which was applied in fish.…”

Impact of biotechnological transgenesis procedures on duck productivity

“…The creation of transgenic poultry is carried out using three main methods: 1) sperm-mediated gene transfer (SMGT) 2) injection of a DNA vector under the embryo cavity of a newly laid egg; 3) introduction of transfected blastomeres into the embryo (Kwon et al, 2018;Konoval et al, 2019;2021). The creation of transgenic poultry by injection is complicated by the lack of transparency of its egg, the presence of a large yolk, and the uniqueness of the reproductive system of the Avis class (Ibrahim & Stadnicka, 2023).…”