Method of differentiation of morula or inner cell mass cells and method of making lineage-defective embryonic stem cells
a technology of inner cell mass and differentiation method, which is applied in the field of differentiation method of morula or inner cell mass cells and the method of making lineage-defective embryonic stem cells, can solve the problems of unstable embryonic stem cell lines, granulosa cells are not easily cultured, and no evidence of development past, so as to prevent the growth of embryonic stem cells
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example 1
[0091] The following example illustrates that only a fraction of the inner cell mass cells cultured are capable of developing into embryonic stem cell-like cells. Thus, there are pluripotent inner cell mass cells which cannot, or do not, develop into embryonic stem cells.
Explant Derivation and Culture
[0092] A. Sample Retrieval [0093] 1) Thoroughly clean the section of tissue to be removed, such as by use of an iodine or ethanol solution. [0094] 2) Remove sample with an ear notcher or scissors and immediately place the tissue in an antibiotic solution (Solution 1 or equivalent). Swirl solution to remove any remaining iodine. [0095] 3) Transfer to a 50 ml conical tube of fresh Solution 1. [0096] 4) Store or ship overnight at 4° C.
[0097] B. Tissue Sectioning and Culture [0098] 1) Remove sample from conical tube and place in fresh warm Solution 1 in a 100 mm culture dish. [0099] 2) Trim any hair using sterile forceps and scissors and transfer to another dish of Solution 1. [0100] 3)...
example 2
[0117] Three adult Holstein steers approximately 8-10 months old (weighing approximately 500-1000 lbs) were purchased from Thomas Morris, Inc., Maryland, and shipped to the South Deerfield Farm at the University of Massachusetts, Amherst. To obtain fibroblasts for nuclear transfer, skin biopsies were obtained from each of the animals by ear notch. A plasmid which expresses a reporter gene encoding enhanced green fluorescent protein (eGFP), was transfected into the cells, and transfected cells were selected with neomycin. Purified cells, analyzed by PCR and / or FISH, were used for nuclear transfer as described previously in Nature (2998) Biotechnol. 16: 642-646, herein incorporated by reference.
[0118] Isolated inner cell mass cells generated from bovine blastocysts are then injected into the paralumbar fascia of the donor steers (three sites with inner mass cells from three 10 day embryos, three sites with inner mass cells from three 12 day embryos, and three sites with inner mass ce...
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