The Ipl (Tssc3) gene lies in an extended imprinted region of distal mouse chromosome 7, which also contains the Igf2 gene. Expression of Ipl is highest in placenta and yolk sac, where its mRNA is derived almost entirely from the maternal allele. Ipl encodes a small cytoplasmic protein with a pleckstrin-homology (PH) domain. We constructed two lines of mice with germ-line deletions of this gene (Ipl neo and Ipl loxP ) and another line deleted for the similar but nonimprinted gene Tih1. All three lines were viable. There was consistent overgrowth of the Ipl-null placentas, with expansion of the spongiotrophoblast. These larger placentas did not confer a fetal growth advantage; fetal size was normal in Ipl nulls with the Ipl neo allele and was decreased slightly in nulls with the Ipl loxP allele. When bred into an Igf2 mutant background, the Ipl deletion partially rescued the placental but not fetal growth deficiency. Neither fetal nor placental growth was affected by deletion of Tih1. These results show a nonredundant function for Ipl in restraining placental growth. The data further indicate that Ipl can act, at least in part, independently of insulin-like growth factor-2 signaling. Thus, genomic imprinting regulates multiple pathways to control placental size. The Ipl gene, also known as Tssc3, lies on distal chromosome 7 of the mouse and human chromosome 11p15.5 (1, 2). This region contains multiple imprinted genes clustered in 1 Mb of DNA. Two of these, p57 Kip2 (Cdkn1c) and Igf2, control fetal and placental growth in mice (3-7) and humans (8). Similar to these genes, Ipl is highly expressed in the extraembryonic tissues (1), but in contrast to these genes, Ipl is expressed only weakly in the embryo proper (1, 9). Ipl encodes a cytoplasmic protein with a pleckstrin-homology (PH) domain (9), thus by analogy with other PH-domain proteins it may modulate cell signaling, intracellular trafficking, or other processes that depend on phosphatidylinositol lipid second messengers. Ipl has two close relatives: TDAG51 and Tih1. Of these genes, Tih1 is most similar to Ipl (9). Tih1 is located in a nonimprinted region of mouse chromosome 1, and it is expressed biallelically (9). To determine the function of Ipl and to accumulate data relevant to the biological rationale for imprinting, we created mice with germ-line deletions of Ipl and Tih1. Materials and MethodsGene Deletions, Genotyping, and Gene Expression. To make the Ipl neo targeting vector, a 5-kb 5Ј genomic NotI restriction fragment and a 6-kb 3Ј EcoRI͞XbaI fragment flanking Ipl were ligated upstream and downstream of Pgk-Neo in pPNT1, yielding pPNT-Ipl. To make the Ipl loxP targeting vector, the Pgk-Neo cassette of pPNT-⌬Ipl was replaced by that from pLNL, which includes flanking loxP sites. To verify homologous recombination, we used genomic PCR products upstream and downstream of the 5Ј and 3Ј cassettes. Recombination of the loxP sites was induced by crossing to HSP-Cre transgenic mice (10). The Ipl deletions (encompassing nucleotides 77,444-78,990 of GenBank a...
Cord blood (CB) natural killer (NK) and lymphokine-activated killer (LAK) cytotoxic cells are poorly characterized but might be used to treat minimal residual and/or recurrent malignant disease. Currently, there is no mechanism to use CB for adoptive cancer cellular immunotherapy after CB transplantation (CBT). Recognizing this as a deficiency, we hypothesized that CB aliquots could be engineered ex vivo for potential donor lymphocyte infusion after CBT. Cryopreserved CB aliquots were thawed, depleted of monocytes, and cultured in serum-free medium alone or serum-free medium with anti-CD3 and interleukins 2, 7, and 12 combined with antibody/cytokines for 48 hours. Immunophenotyping, cytotoxicity, and proliferation were evaluated. A significant expansion of CD3+ was seen, in addition to increases in lymphocyte subsets of CD8+, CD8+/CD25+, and CD3+/45RO+ versus medium alone. A significant enhancement of CD3 proliferation (P<.001), NK cytotoxicity, NK subset expansion, LAK cytotoxicity, and T-helper 1 subset expansion was also demonstrated. Significant enrichment was seen in NK CD16+/CD56+bright, CD16+/CD56+dim, CD56+bright and CD56+dim/KIR3DL1+, CD56+bright and CD56+dim/KIR2DL1+, CD56+bright and CD56+dim/KIR2DL2+ and CD94+/NKG2a+ subsets. These increases in CB NK subsets were in part secondary to augmentation of cell survival. Further, survival of NOD-SCID mice xenografted with human K562 cells and treated with CB cells expanded with antibody/cytokines was significantly higher than that in animals that received no treatment (phosphate buffered saline) and those that were treated with CB ex vivo expanded in medium alone (P<.005, respectively). These data suggest that cryopreserved CB cells could be ex vivo engineered for potential use as adoptive cancer cellular immunotherapy for donor lymphocyte infusion after CBT.
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