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Method of producing non-human mammals

a technology of non-human mammals and a method of production, applied in the field of non-human mammals, can solve the problems of requiring considerable time and resources, producing desired genetically modified mammals, etc., and achieve the effects of reducing the extent or duration of the condition, and reducing the extent or severity

Inactive Publication Date: 2011-03-10
WHITEHEAD INST FOR BIOMEDICAL RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach significantly reduces the time and resources required to generate mutant mammals by eliminating the need for breeding chimeric intermediates, facilitating the production of animals with multiple genetic alterations and allowing for the derivation of fertile XO females and genetically identical offspring without outcrossing.

Problems solved by technology

Producing desired genetically manipulated mammals, even those for which the breeding cycle is relatively brief, requires considerable time, as well as resources, using current methods.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Assessment of the Effects of Genetic Heterogeneity of Donor Cells on Development of ES Cell-Tetraploid Pups

[0038]The possible effect of genetic heterogeneity of the donor cells on the development of ES cell-tetraploid pups was tested by transferring inbred or F1 ES cells into tetraploid blastocysts and assessing survival. Injection of ES cells into the blastocoel cavity of tetraploid blastocysts was aided by the use of a piezo-driven micromanipulator and the resulting composite embryos were transferred to recipient females. 312 tetraploid blastocysts were injected with four different inbred ES cell lines that gave rise to 20 pups (6%) that were alive and active at cesarean section. However, 17 of the 20 newborns died of respiratory failure within 30 minutes. Of the three remaining pups, two were unable to sustain respiration and died within the next few hours (Table 1). Only one inbred ES cell-tetraploid pup was able to sustain respiration and developed to adulthood. In contrast, of...

example 2

Histological Assessment of Lungs

[0040]ES cell-tetraploid pups derived from inbred ES cells appeared to suffer from respiratory distress after delivery. Histological analysis of both inbred and F1 completely ES cell derived neonates was carried out. Examination of the lungs from inbred ES cell-tetraploid pups revealed that the alveoli were not inflated, while the lungs of newborns derived from F1 ES cells were fully inflated. In addition, interstitial bleeding was often seen in inbred ES cell derived mice. These observations suggest that the failure to initiate breathing and / or sustain normal circulation likely contributed to postnatal death of inbred ES cell-tetraploid pups.

[0041]Results described herein demonstrate that genetic heterozygosity is a crucial parameter influencing postnatal survival of pups derived from ES cells by tetraploid embryo complementation. Pups derived from inbred ES cells die perinatally with a phenotype of respiratory failure. In contrast, the great majorit...

example 3

Generation of Fertile Mice Carrying A Mutation of Interest

[0044]A male targeted F1 ES cell line was made as described above. The cell line was then screened by Southern blot for subclones that have lost the Y chromosome. The probe used in the Southern blot to identify targeted (mutant) ES subclones which have lost the Y chromosome was a Y chromosome probe against repetitive elements. This probe was previously described by Lamar, E. E. and Palmer, E., Cell, 37:171-177 (1984).

[0045]Subclones that have lost the Y chromosome were taken and used to make mice by tetraploid embryo complementation as described above. The female ES mice produced were shown to be fertile, indicating that they can transmit the mutation of interest.

[0046]Male ES mice were produced from parent cell lines as described above. The male ES mice produced were shown to be fertile.

[0047]The loss of the Y chromosome, as shown by Southern blot analysis, is a general phenomenon in several ES lines, as demonstrated by the ...

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Abstract

A method of producing mutant / targeted non-human mammals, such as mutant mice that does not require production of chimera and permits the introduction of multiple mutations in embryos and, thus, avoids the necessity of breeding to combine all of the desired mutations in a single animal. The method is efficient in producing ES mice.

Description

RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 10 / 850,344, filed May 19, 2004, which is a continuation of U.S. application Ser. No. 09 / 957,659, filed Sep. 20, 2001 (now abandoned), which is a continuation-in-part of U.S. Application Ser. No. 09 / 755,003, filed Jan. 5, 2001 (now U.S. Pat. No. 6,784,336), which claims the benefit of the filing date of U.S. Provisional Application No. 60 / 234,378, filed Sep. 20, 2000 and the filing date of U.S. Provisional Application No. 60 / 255,970, filed Dec. 15, 2000. This application is also related to U.S. application Ser. No. 10 / 787,847, filed Feb. 26, 2004 (now abandoned). The entire teachings of the referenced applications are incorporated herein by reference.GOVERNMENT SUPPORT[0002]Work described herein was supported, in whole or in part, by National Institutes of Health Grants No. 5-R35-CA44339 and RO1-CA84198. The United States government has certain rights in the invention.BACKGROUND OF THE INVENTION...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027C12Q1/04C12Q1/68C12N15/00A61B17/43C12N15/06C12N15/873
CPCA01K67/027A01K2217/05C12N2517/02C12N15/873C12N2510/00A01K2217/075
Inventor EGGAN, KEVIN C.JAENISCH, RUDOLF
Owner WHITEHEAD INST FOR BIOMEDICAL RES
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