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Compositions and methods relating to tumor analysis

A tumor, tumor cell technology, applied in the field of compositions and methods related to tumor analysis, which can solve problems such as cancer model limitations

Inactive Publication Date: 2018-08-31
JACKSON LAB THE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most cancer models used to study and evaluate cancer and new drugs consist of in vitro cell lines, and given the complexity of physiological processes in vivo, analysis of such in vitro models may be of limited value
Current in vivo cancer models are often limited in application, especially where analyzes need to be performed as quickly as possible (e.g. analysis of patient-derived tumor cells in xenograft tumors grown in vivo)

Method used

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  • Compositions and methods relating to tumor analysis
  • Compositions and methods relating to tumor analysis
  • Compositions and methods relating to tumor analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0178] NSG-BALDP159L mice carrying a point mutation p.P159L in the Rhbdf2 gene were generated using CRISPR / Cas9 technology. Microinjection of Cas9 mRNA, truncated guide RNA (sgRNA), and single-stranded oligonucleotide DNA (ssDNA:

[0179] ) to target the Rhbdf2 locus in NSG embryos to mutate the proline at amino acid 159 of SEQ ID NO:1 to leucine. The underlined codon CTA is a mutated codon introduced into the genome. figure 1 A schematic diagram of mouse iRhom2 (SEQ ID NO: 1 ) is shown.

[0180] sgRNA used to generate mouse (17mer): G CAG ATT GTG GAT CCAC (SEQ ID NO: 7)

[0181] CRISPR / Cas9 protocol:

[0182] 1. Turn on the PCR instrument and centrifuge - close the lid and set the temperature to 4°C.

[0183] 2. RNAase-Zap (working area).

[0184] 3. Remove the following from -80°C to ice (in a labeled box directly in front of the bottom rack)

[0185] a.sgRNA

[0186] b. Cas9 mRNA

[0187] 4. Centrifuge guide RNA and Cas9 mRNA at 20,000x g for 15 minutes at 4°C

...

Embodiment 2

[0235] animal

[0236] Six to eight week old NSG-BALDP159L (n=5) and NSG female mice (n=4) were used to examine tumor growth. Mice were bred and maintained under specific pathogen free (SPF) conditions at the Jackson Laboratory. Food and acidified water were provided ad libitum.

[0237] Xenogeneic tumor cell preparation and administration

[0238] MDA-MB 231 human breast cancer cells were cultured in RPMI medium and grown at 37 °C. MDA-MB 231 human breast cancer cells (3×10 6 ) were injected subcutaneously into NSG-BALDP159L and NSG mice.

[0239] tumor size

[0240] The subcutaneous xenograft tumor diameter was measured daily using an external caliper. To determine tumor volume by external calipers, the maximum longitudinal diameter (length) and the maximum transverse diameter (width) were measured using external calipers. Then the tumor volume is calculated as: 1 / 2 (length × width 2 ) = tumor volume. At the end of the study, tumors were collected and subjected to h...

Embodiment 3

[0249] Such as comparing SEQ ID NO:1 and 3 Figure 6 As shown, mouse iRhom1 and mouse iRhom2 are highly related proteins. Given the high structural identity of mouse iRhom1 and mouse iRhom2, studies were performed to determine whether the functional effects of modifications in iRhoml were similar to those observed for iRhom2 modifications.

[0250] generated Rhbdf1 knockout C57BL / 6 mice, generated iRhom1-deficient mice, and found that iRhom1 deficiency resulted in weight loss. Figure 7 showed a normal-sized Rhbdf1 gene deletion heterozygous (Rhbdf1+ / - ) mice (right) homozygous for the deletion of the Rhbdf1 gene (Rhbdf1 - / - ) size differences between mice (left).

[0251] Rhbdf1 knockout C57BL / 6 mice died at 3–4 weeks of age, as Figure 8 shown in Fig. Rhbdf1 gene deletion heterozygous (Rhbdf1 + / - ) mice showed a normal survival percentage ( Figure 8 upper line in the middle panel), and are viable and fertile, whereas Rhbdf1 gene deletion homozygous (Rhbdf1 - / - ) mice...

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Abstract

A genetically modified NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ mouse is provided by the present invention wherein the genome of the mouse includes a mutated Rhbdf2 gene such that the mouse expresses a mutant iRhom2 protein, wherein the mutant iRhom2 protein differs from wild-type iRhom2 protein due to one or more mutations selected from p.I156T, p.D158N and p.P159L, and wherein the mouse is characterized by hairless phenotype and increased growth of an exogenous tumor compared to a mouse of the same genetic background which express wild-type iRhom2 protein.

Description

[0001] Cross references to related patent applications [0002] This application claims priority to US Provisional Patent Application No. 62 / 248,417, filed October 30, 2015, which is incorporated herein by reference in its entirety. technical field [0003] According to a specific aspect, the invention provides a genetically modified NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mouse, wherein the genome of the mouse includes a mutated Rhbdf2 gene such that the mouse expresses a mutated iRhom2 protein, wherein the mutated iRhom2 protein is due to one or more selected from p.I156T , p.D158N and p.P159L mutations that differ from the wild-type iRhom2 protein, and wherein the mice are characterized by a hairless phenotype and increased Exogenous tumor growth. Background technique [0004] Although significant advances in the diagnosis and treatment of cancer have been made in recent years, the disease remains common and widespread. According to the National Cancer Institute's S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12N9/64G01N33/50A61K49/00
CPCA01K67/0275A01K67/0278A61K49/0008C12N9/6424G01N33/5011G01N33/5088A01K2207/12A01K2217/072A01K2217/15A01K2227/105A01K2267/0331A01K67/0276A01K2217/075G01N33/502
Inventor M·V·怀尔斯V·霍苏尔L·D·舒尔茨
Owner JACKSON LAB THE
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