A method of generating sterile and monosex progeny

A fertile, female technology for the production of sterile and parthenogenetic progeny that addresses increased operating costs, increased disease susceptibility, and increased mortality in sterile organisms

Pending Publication Date: 2021-07-23
センター フォー アクアカルチャー テクノロジーズインコーポレイティド
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] One or more previously proposed methods of sterilizing freshwater and marine organisms may result in: (1) insufficient efficacy; (2) increased difficulty in reproducing the sterility trait, e.g., genetic selection must be performed to identify subpopulations of sterile individuals , and/or repeated treatments per generation; (3) increased operating costs, e.g., implementing major changes in ...

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  • A method of generating sterile and monosex progeny
  • A method of generating sterile and monosex progeny
  • A method of generating sterile and monosex progeny

Examples

Experimental program
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Effect test

Embodiment 1

[0106] Example 1 - Materials and methods

[0107] Animals used and ethics statement: All experiments complied with US regulations, ensuring that animal welfare and husbandry procedures were performed in accordance with the IACUC-approved animal protocol CAT-004. The tilapia (Oreochromis niloticus) strain used in this study was derived from a Brazilian strain obtained from a commercial producer in the United States.

[0108] Nuclease Generation and Its Strategy: Generation of F0 mutants: homologous gene lines of cyp17, Cyp19a1a, Tjp1a, Csnk2a2, Hiat1, Smap2, Gopc, Gsdf, Dmrt1, FSHR and vitellogenin genes (VtgAa and VtgAb) of tilapia were obtained from genome databases by computer Identification.

[0109] To generate DNA double-strand breaks (DSBs) at specific genomic loci, we used engineered nucleases. In most applications, a single DSB is generated in the absence of a repair template, resulting in activation of the non-homologous end-joining (NHEJ) repair pathway. NHEJ ...

Embodiment 2

[0126] Example 2 - Using gene editing tools to induce biallelic knockout in the F0 generation of tilapia

[0127] We independently targeted two genes involved in pigmentation, the gene encoding tyrosinase (tyr) [2] and the mitochondrial inner membrane protein MpV17 (MpV17) (Krauss, Astrinides et al. 2013) [8] . We found that 50% and 46% of the injected embryos showed hypermutation at the tyr and mpv17 loci, respectively ( Figure 4 ). Loss of the cell-function allele autonomously leads to the embryonic body ( Figure 4 B) and retinal pigment epithelium ( Figure 4 The achromatic cell mass in C) produces embryonic phenotypes ranging from complete loss of melanin to partial loss and iris pigmentation that are easily differentiated from wild-type phenotypes ( Figure 4 A and C). Embryos showing complete lack of pigmentation (10-30% of treated fish) were reared to 3 months of age, all lacking wild type tyr and mpv17 sequences. These fish display transparent and albino phenot...

Embodiment 3

[0128] Example 3 - Multigene Targeting in Tilapia

[0129] We tested whether multiple loci in the tilapia genome could be targeted simultaneously and whether mutagenesis efficiency measured at one locus could predict mutations at other loci. To test our hypothesis, we co-targeted tyr and dead-end 1 (dnd). Dnd is a PGC-specific RNA-binding protein (RBP) that maintains germ cell fate and migration ability [3]. After injection of programmed nuclease, we found that mutations in two gene targets, Tyr and Dnd, were highly correlated. Approximately 95% of abino(tyr) mutants also carry mutations at the dnd locus, demonstrating the suitability of pigmentation defects as selectable markers ( Figure 5 A). After further analysis of the gonads of 10 albino fish, 6 had translucent agenic testes ( Figure 5 B). Vasa, a germ cell-specific marker strongly expressed in wild-type testis, was not significantly expressed in dnd mutant testis. This result suggests that expression of zygotic ...

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Abstract

The disclosure provides a method of generating a sterile sex-determined fish, crustacean, or mollusk. The method comprises breeding (i) a fertile homozygous mutated female fish, crustacean, or mollusk having at least a first mutation and a second mutation with (ii) a fertile homozygous mutated male fish, crustacean, or mollusk having at least the first mutation and the second mutation to produce the sterile sex-determined fish, crustacean, or mollusk. The first mutation disrupts one or more genes that specify sexual differentiation, the second mutation disrupts one or more genes that specify gamete function, and the fertility of the fertile homozygous female fish, crustacean, or mollusk and the fertile homozygous mutated male fish, crustacean, or mollusk has been rescued. The disclosure also provides methods of making broodstock for use in producing sterile sex-determined fish, crustacean, or mollusks, as well as the broodstock itself.

Description

[0001] Statement of Government Rights [0002] Aspects of the work described in this paper were supported by a grant 2018-33522-28745 from the USDA National Institute of Food and Agriculture. The U.S. Government may have certain rights in these inventions. technical field [0003] The present invention generally relates to methods of sterilizing and sexing freshwater and marine organisms. Background technique [0004] The following paragraphs are not an admission that anything discussed therein is prior art or part of the knowledge of those skilled in the art. [0005] Fish have been genetically engineered (GE) modified to produce valuable medicinal proteins or to incorporate beneficial properties for aquaculture. To meet future demand for seafood and to increase the sustainability of the aquaculture industry, various fish species have been bred with improved growth rates, food conversion rates, disease resistance, and nutritional value. However, worldwide adoption of the...

Claims

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Application Information

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IPC IPC(8): A01K67/027A01K67/033
CPCA01K67/0276A01K67/0275A01K2207/12A01K2217/075A01K2227/40A01K2267/02C07K14/461Y02A40/81A01K67/027A01K67/033A01K67/0333A01K2207/20A01K2227/70
Inventor X·C·劳斯J·T·布坎南
Owner センター フォー アクアカルチャー テクノロジーズインコーポレイティド
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