Gene Targeting and Patholgogy Core - Children's Hospital Los Angeles

Summary

The purpose of the Genome Targeting and Pathology Core is to provide genetically manipulated mice for biomedical studies, including:

• Transgenics
• Knockins
• Conditional/tissue specific knockouts
• Providing investigators with centralized support for the performance of murine tissue preparation for histological analysis

 

Equipment/Services

Services

The Gene Targeting and Pathology Core provides the following services:

• Conditional knockouts, knockins & lentiviral transgenesis
  Lentiviral transgenesis is used to produce transgenic mice by introducing a transgene into mouse morulae by lentiviral gene transfer.
  • The user provides prepared lentiviruses (purification required) along with a plan for genotyping. The Gene Targeting and Pathology Core prepares morulae, transduces morulae and expands them to blastulas. The core then implants expanded blastulas into foster females and keeps resulting pups until tail biopsies are performed by a client (within 4 weeks). The user breeds positive transgenics for germ-line transmission.
• Conditional/Tissue-specific knockouts and knockins
  Site-specific mutation of a specific locus is inserted in the gene of interest using homologous recombination in mouse embryonic stem cells, and subsequent generation of chimeras and germline transmitters.
  • Outline for the production of HR ES cells:
    After consultation with the Gene Targeting and Pathology Core, the user designs a targeting vector and screenings strategy and then creates a targeting vector. The user then confirms the practicality of screening strategy and provides it to the core. The user purifies DNA according to a protocol provided by the core and the core prepares ES cells and electroporates DNA. The core cultures, drug selects ES cells and selects the ES cell clones. The user prepares and screens ES cell DNA and the core prepares cells for injection.
  • Outline for the injection of blastocysts to generate knockout mice:
    Either the user or the core provides the prepared ES cells (note: user provided cells require mycoplasma test results). The core prepares blastocysts and injects 1 or 2 clones of ES cells. The core implants injected blastocysts into foster mice.
• Tissue paraffin embedding and sectioning
  
• Routine H & E staining
  
• Genotyping of tail DNA

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Equipment and Software

Nikon Eclipse E600 Microscope
Narishige IM5B Microinjection x2
Narishige MO202 Micromanipulator x2
Leica Microscope MZ95
Leica Light Source KL1500LCD
Leica Microtome RM2135
Leica Microtome RM2235
Leica Embedding Station EG1160
Leica Microscope 200M100
Leica Vibrotome VT1000S
Leica Microscope DM1 3000B
Hera Cell Incubator CELL 150 x3
Fisher Scientific Microscope 12-560-45
Precision Water Bath 183
Steril Gard III Advance Class II Biosafety Cabinet from Baker
Frigidaire Refrigerator Model GLRT13TEWI
Leica Water Bath HI1210
Leica Power Source NCL 150
Thermo Forma Liquid Nitrogen Tank Model 740
Leica EG 1150 Embedding Station
Leica RM 2125 Microtome
BTX Electroporator ECM 830

Price List

Fee Structure

Gene Targeting & Pathology Core Service	Charge
Embedding – Self service	$3.5/ea
Embedding – Core service	$12/ea
Microtome – Self Service	$3/hr
Microtome – Core service	$3/slide
H&E Staining – Core service	$10/slide
Mouse Line Derivation	$750
Lenti-viral Transgensis	$2500
ES cell culture	$4000
Blastocyst Injection	$4000
Consultation	$50/hr

Turn Around Time

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Abstract

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More information

Website

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Location

Childrens Hospital Los Angeles Saban Research Building, Room 544

Contact Information

Silverio Bellusci, PhD Director E-mail: sbellusci@chla.usc.edu

Scheduling Appointments

Contact Silverio Bellusci, PhD at sbellusci@chla.usc.edu