Haploid cells

A haploid cell, haploid technique, applied in the field of newly identified genes

Inactive Publication Date: 2014-09-03
IMBA INSTITUT FUR MOLEKULARE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of this, means and methods allowing a high-throughput approach to recessive genetics in mammals would be highly desirable, but not yet available

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0213] [Example 1: Method]

Embodiment 11

[0214] [Example 1.1: Parthenogenetic Derivation of Haploid Stem Cells]

[0215] C57BL / 6 x 129F1 female mice were superovulated using standard procedures, and unfertilized oocytes were washed and collected. For activation, oocytes were exposed to 5% ethanol or 25 mM SrCl as described 2 (Kaufman et al., 1983; Otaegui et al., 1999). After 4 hours of activation, viable oocytes were transferred into pseudopregnant 129 females, recollected at embryonic day 3.5 (ED), and cells were derived according to established embryonic stem cell derivation protocols (Bryja et al., 2006). Parthenogenetically derived stem cells are initially maintained on feeder layers and subsequently adapted to feeder-free culture conditions. Haploid cells are trained to grow under feeder-free conditions by gradually reducing the feeder cell density and eventually removing the feeder cells from the culture. Stem cell medium consisted of DMEM with 15% FCS (Gibco) supplemented with 2 mM L-glutamate, 1 mM sodium...

Embodiment 12

[0216] [Example 1.2: Genome coverage analysis and SNP mapping]

[0217] Genomic DNA preparations were sheared, adapters ligated and subjected to Illumina sequencing (HiSeq) by using a Covaris DNA sonicator according to the manufacturer's protocol. Only the Unique genome coordinates. SNPs, which differ between C57BL / 6 and 129, were retrieved from Sanger (www.sanger.ac.uk / resources / mouse / genomes / ) and mismatches observed during genomic mapping of deep sequencing reads against them were evaluated. Each SNP covered by Solexa reads was assigned to C57BL / 6 and 129 based on the majority of reads.

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Abstract

The present invention relates to the generation of stable haploid cell cultures, uses of said cells in forward and reverse genetics, especially the identification of target genes associated with a modified phenotype and in particular identifying genetic targets associated with toxin resistance, especially ricin toxicity resistance, and therapeutic uses of target compounds.

Description

【Technical field】 [0001] The present invention relates to haploid cells and their use as genetic screening tools, and newly identified genes associated with cell survival and toxin resistance. 【Background technique】 [0002] Diploid genomes of complex organisms severely limit many genetic approaches in the study of model species, but also in humans. Some organisms, such as yeast or social insects, are haploid, ie they carry a single set of chromosomes (Otto and Jarne, 2001). Haploidy in yeast has been exploited to identify fundamental mechanisms of biology (Hartwell et al., 1974). However, all somatic mammalian cells carry two copies of chromosomes, ie, exhibit diploidy that obscures mutation screening. Organisms with a single copy of their genome, such as yeast, provide the basis for genetic analysis in which any recessive mutation in an essential gene will reveal a clear phenotype due to loss of a second gene copy (Hartwell et al., loc.cit .). It has recently been show...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735G01N33/50
CPCC12N2503/02C12Q1/686C12N5/0606C12Q2600/106G01N33/5014C12Q1/025A61P31/00A61P31/04
Inventor U·艾林J·彭宁格J·陶贝茨米德
Owner IMBA INSTITUT FUR MOLEKULARE BIOTECH
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