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Methods for gender determination of avian embryos in unhatched eggs and means thereof

A poultry and gender technology, applied in biochemical equipment and methods, other methods of inserting foreign genetic materials, microbe determination/inspection, etc., can solve problems such as effective and non-invasive methods without sex identification

Inactive Publication Date: 2018-08-31
EGGXYT LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0031] Therefore, there is currently no effective and non-invasive method of sexing chickens at the egg stage prior to hatching available

Method used

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  • Methods for gender determination of avian embryos in unhatched eggs and means thereof
  • Methods for gender determination of avian embryos in unhatched eggs and means thereof
  • Methods for gender determination of avian embryos in unhatched eggs and means thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0251] Selection of reporter genes for visual sex recognition in poultry

[0252] To demonstrate the feasibility of visually identifying the sex of poultry in ovo, the application of bioluminescence was evaluated in comparison to a fluorescent reporter. Therefore, a transgenic mouse expressing a reporter gene, such as firefly luciferase (having a nucleic acid sequence as shown in SEQ ID NO: 20; encoding an amino acid sequence as shown in SEQ ID NO: 21) and Green fluorescent protein (eGFP).

[0253] To observe luciferase activity, luciferin was injected subcutaneously into luciferase-expressing transgenic mice in Figure 1, then the tail and ears were excised and introduced through a 5 mm hole in the eggshell of unfertilized eggs. As shown in the figure, in tail and ear samples ( Figure 1A , 1B) A luciferase detectable signal was clearly observed through the eggshell. Therefore, the present inventors next examined the feasibility of inducing a luciferase response in ovo. Ther...

Embodiment 2

[0259] Design of guide RNA vector

[0260] To integrate the luciferase reporter gene in sex chromosome W or Z, a CRISPR / Cas9-mediated HDR approach was chosen. The relevant gRNA loci were then searched from the two sex chromosomes.

[0261] The region 1022859-1024215 of chromosome W of a female chicken containing the nucleic acid sequence shown in SEQ ID NO.3 was analyzed for guide RNA design. Through the non-restricted cloning scheme, select, synthesize two guide RNAs, and clone it into the Cas9 SmartNuclease carrier containing wild-type Cas9 nuclease (Horizon), respectively, the two guide RNAs are: gRNA1:GCACTAGGAACCAGCAGCAG, as SEQ ID Shown in NO.1, and gRNA2: GTAGCCCCAAGAGGGCTAGG, shown in SEQ ID NO.2.

[0262] The predicted parameters for these two gRNAs are shown in Table 1:

[0263] Table 1

[0264] gRNA parameters

gRNA1

gRNA2

sgRNA Designer

0.506

0.63

sscore

0.8677

0.5323

sgRNA scorer

94.8

99.9

[0265] F...

Embodiment 3

[0269] Design of Luciferase Targeting Vector

[0270] Flanking sequences homologous to the appropriate flanking sequences described above for the female W chromosome or female Z chromosome loci are introduced upstream of the CMV promoter and downstream of neomycin resistance or polyA sites in vectors expressing luciferase (Custom synthetic DNA, Integrated DNA Technologies, Inc., USA).

[0271] For the female W chromosome, the reporter gene, especially luciferase, can be cloned using Guide 1 (gRNA1) shown in SEQ ID NO.1 or Guide 2 (gRNA2) shown in SEQ ID NO.2. For cloning using gRNA1, a "left arm" comprising the nucleic acid sequence shown in SEQ ID NO.4 and a "right arm" comprising the nucleic acid sequence shown in SEQ ID NO.5 are provided. For cloning using gRNA2, a "left arm" comprising the nucleic acid sequence shown in SEQ ID NO.6 and a "right arm" comprising the nucleic acid sequence shown in SEQ ID NO.7 are provided.

[0272] Further, the "left arm" of the region upst...

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Abstract

The present invention relates to methods of gender determination and identification in avian subjects. More specifically, the invention provides non-invasive methods using transgenic avian animals that comprise at least one reporter gene integrated into at least one gender chromosome Z or W. The transgenic avian animals of the invention are used for gender determination and selection of embryos inunhatched avian eggs.

Description

technical field [0001] The present invention relates to methods of sex determination and identification in avian subjects. More specifically, the present invention provides non-invasive methods and transgenic avian animals for sex determination and selection of embryos in unhatched avian eggs. Background technique [0002] References considered to be relevant to the subject matter of the present disclosure are listed below: [0003] · WO 2010 / 103111 [0004] ·WO 2014 / 0296707 [0005] ·US 6244214 [0006] ·06124456A2 [0007] ·US2014069336A [0008] ·WO16005539 [0009] ·WO 96 / 39505 [0010] ·WO 97 / 49806 [0011] ·Quansah, E., Long, J.A., Donovan, D.M., Becker, S.C., Telugu, B., FosterFrey, J.A., Urwin, N. 2014. Sperm-mediated transgenesis in chicken using a PiggyBac transposon system. Poultry Science Association Meeting Abstract. BARCPoster Day. [0012] · Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., & Charpentier, E. (2012). A programmable dual-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6879A01K67/027C12N5/10C12N15/85C12N15/90
CPCC12Q1/68C12N9/0069C12N15/102C12N15/907A01K2217/07A01K2227/30A01K2267/0393C12Q1/6879C12Q1/66A01K67/0275C12N15/113C12N2310/20
Inventor D·奥芬
Owner EGGXYT LTD
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