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Selection of pluripotent cells for production of fertile xy female mice

A pluripotent cell, female technology, applied in the fields of genetics and histology and embryology, can solve expensive and time-consuming problems

Active Publication Date: 2018-10-23
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mating requirement implies an expensive and time-consuming step

Method used

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  • Selection of pluripotent cells for production of fertile xy female mice
  • Selection of pluripotent cells for production of fertile xy female mice
  • Selection of pluripotent cells for production of fertile xy female mice

Examples

Experimental program
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Embodiment 1

[0452] Example 1. Generation of fertile and fecund F0 XY female mice from XY ES cells Materials and methods

[0453] ES cell culture: All results described here are from experiments performed with VGF1, our C57BL 6NTac / 129S6SvEv F1 hybrid XY ES cell line (Poueymirou et al. (2007) Nat. Biotechnol. 25:91-99; Valenzuela et al. (2003) Nat . Biotechnol. 21:652-659). ES cells were cultured as previously described (Matise et al. (2000) Production of targeted embryonic stem cell clones. Joyner AL (eds.) Gene targeting: a practical aproach. Oxford University press, New York, pp. 101-132). with NaHCO indicated in Table 3 3 Concentrations of customized high glucose DMEM medium (LifeTech) supplemented with 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 4 mM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin ( Life Technologies), 15% FBS (Hyclone) and 2,000 U / ml Leukemia Inhibitory Factor (LIF, Millipore). The osmolality of the medium was ...

Embodiment 2

[0494] Example 2. In embryos derived from XY ES cells cultured in low osmolality medium Expression of Ddx3y(Dby), Eif2s3y and Sry was silenced or reduced

[0495] Embryos at the developmental stage 11.5 days postcoitus (dpc) were dissected, using Thiele staging to determine their approximate stage. Because gestational age varies widely within a litter, more precise staging of individual embryos was achieved by counting the number of tail somites (ts) from the posterior of the hindlimb bud to the end of the tail. Using this technique, embryos with 8 caudal somites are 10.5 dpc, 18 caudal somites are 11.5 dpc, and 30 caudal somites are 12.5 dpc (according to Hacker, Capel, Goodfellow and Lovell-Badge, Devel 1995).

[0496] In B6 mice, Sry was reported to be detectable at about 13ts, peak at about 17-18ts, and decline by about 27ts. Embryos with ts between 12-27 were obtained from mice to capture the complete time course of Sry. The genital ridge (containing the gonads and ...

Embodiment 3

[0525] Ddx3y (Dby), Eif2s3y and Uty expression in embodiment 3.XY ES cell clones lack or reduce and from there These XY ES cell clones produce fertile XY F0 female mice associated

[0526] Table 11 shows that XY ES cell clones obtained from gene targeting in the VGF1 ES cell line produced females after microinjection into 8-cell embryos Propensity. All clones were maintained and cultured in KO-DMEM-based media throughout the targeting and microinjection process. From this clonal sample, it is evident that XY females are produced The capacity is clone-specific and ranges from 0-60%. These results were reproducible in multiple microinjection experiments: non-female-producing clones maintained the trait across repeated microinjections, while female-producing clones tended to produce approximately the same proportion of females in repeated microinjection experiments . The effect was not reversible by regrowth of the clones in DMEM-based media. Gene targeting from whic...

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Abstract

Methods and compositions are provided for generating F0 fertile XY female animals. The methods and compositions involve making XY pluripotent or totipotent animal cells, in vitro cell cultures, or embryos that are capable of producing a fertile female XY animal in an F0 generation. Such cells, embryos, and animals can be made by silencing a region of the Y chromosome. Optionally, the cells can also be cultured in feminizing medium such as a low-osmolality medium and / or can be modified to decrease the level and / or activity of a Sry protein. Methods and compositions are also provided for silencing a region of the Y chromosome in an XY pluripotent or totipotent animal cell, or in vitro cell cultures, embryos, or animals derived therefrom, by maintaining an XY pluripotent or totipotent animalcell in a feminizing medium. Methods and compositions are also provided for maintaining a population of XY pluripotent or totipotent animal cells in a feminizing medium and selecting cells or clones having increased capabilities for producing a fertile female XY animal in an F0 generation. Methods and compositions are also provided for screening for compounds with feminizing activity or for optimizing concentrations of components in feminizing media.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Application No. 62 / 219,927, filed September 17, 2015, which is hereby incorporated by reference in its entirety for all purposes. [0003] References to Sequence Listings Submitted as Text Files via EFSNet [0004] The sequence listing written in file 484387SEQLIST.txt is 9.17 kb, was created on September 16, 2016, and is hereby incorporated by reference. technical field [0005] The present invention relates to the fields of genetics and histology and embryology, in particular to methods and compositions for producing F0 fertile XY female animals. Background technique [0006] Generation of genetically modified mice carrying targeted mutations is usually achieved with XY ES cell lines derived from male embryos. Injection of ES cells into blastocyst-stage embryos, which may be male or female, followed by uterine transfer into a surrogate mother results in the birth of chim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12N5/0735
CPCA01K2207/35A01K2267/02A01K67/0271A01K2207/12A01K2227/105C12N5/0606C12Q2600/158C12Q2600/136C12Q2600/156G01N33/5073C12Q1/6876A01K67/027
Inventor 詹尼弗·施马尔大卫·佛伦杜威琼科·库诺家珍·萧古斯塔沃·德罗格特瑜·白沃基特克·奥尔巴赫
Owner REGENERON PHARM INC
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