Biomedical subjects

Pentao Liu

Publications and source records attributed to Pentao Liu.

4 recordsLinked to original sources

PubMed2003

Bcl11a is essential for normal lymphoid development.

Bcl11a (also called Evi9) functions as a myeloid or B cell proto-oncogene in mice and humans, respectively. Here we show that Bcl11a is essential for postnatal development and normal lymphopoiesis. Bcl11a mutant embryos lack B cells and have alterations in several types of T cells. Phenotypic and expression studies show that Bcl11a functions upstream of the transcription factors Ebf1 and Pax5 in the B cell pathway. Transplantation studies show that these defects in Bcl11a mutant mice are intrinsic to fetal liver precursor cells. Mice transplanted with Bcl11a-deficient cells died from T cell leukemia derived from the host. Thus, Bcl11a may also function as a non-autonomous T cell tumor suppressor gene.

Animals↗

PubMed2003

A highly efficient recombineering-based method for generating conditional knockout mutations.

Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. Here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations. This method uses homologous recombination mediated by the lambda phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce loxP or FRT sites into the subcloned DNA. Unlike other methods that use short 45-55-bp regions of homology for recombineering, our method uses much longer regions of homology. We also make use of several new E. coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive lambda prophage, and the Cre or Flpe recombinases from an arabinose-inducible promoter. We also describe two new Neo selection cassettes that work well in both E. coli and mouse ES cells. Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in less than 2 wk. This method should also facilitate the generation of knock-in mutations and transgene constructs, as well as expedite the analysis of regulatory elements and functional domains in or near genes.

Animals↗

PubMed2002

Evi3, a common retroviral integration site in murine B-cell lymphoma, encodes an EBFAZ-related Krüppel-like zinc finger protein.

Retroviral insertional mutagenesis in inbred mouse strains provides a powerful method for cancer gene discovery. Here, we show that a common retroviral integration site (RIS) in AKXD B-cell lymphomas, termed Evi3, encodes a novel zinc finger protein with 30 Krüppel-like zinc finger repeats. Most integrations at Evi3 are located upstream of the first translated exon and result in 3' long-terminal repeat (LTR)-driven overexpression of Evi3. Evi3 is highly related to the early B-cell factor-associated zinc finger gene (Ebfaz), and all 30 zinc fingers found in EVI3 are conserved in EBFAZ. EBFAZ binds to and negatively regulates early B-cell factor (EBF) (also known as olfactory-1, OLF1), a basic helix-loop-helix (bHLH) transcription factor required for B-lineage commitment and the development of the olfactory epithelium. EBFAZ also binds to SMA- and MAD-related protein-1 (SMAD1) and SMAD4 in response to bone morphogenetic protein-2 (BMP2) signaling, which in turn activates the homeobox regulator of Xenopus mesoderm and neural development Xvent-2. Surprisingly, while Ebfaz and Evi3 are coexpressed in many tissues, and both proteins are nuclear, we could not detect Ebfaz expression in B cells by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas Evi3 expression could be detected at all stages of B-cell development. Our results suggest that EVI3, like EBFAZ, is a multifunctional protein that participates in many signaling pathways via its multiple zinc fingers. Furthermore, our results suggest that EVI3, not EBFAZ, is the member of this protein family that interacts with and regulates EBF in B cells.

Amino Acid Sequence↗

PubMed2001

Efficient Cre-loxP-induced mitotic recombination in mouse embryonic stem cells.

FLP/FRT-induced mitotic recombination provides a powerful method for creating genetic mosaics in Drosophila and for discerning the function of recessive genes in a heterozygous individual. Here we show that mitotic recombination can be reproducibly induced in mouse embryonic stem (ES) cells, by Cre/loxP technology, at frequencies ranging from 4.2 x 10(-5) (Snrpn) to 7.0 x 10(-3) (D7Mit178) for single allelic loxP sites, and to 5.0 x 10(-2) (D7Mit178) for multiple allelic lox sites, after transient Cre expression. Notably, much of the recombination occurs in G2 and is followed by X segregation, where the recombinant chromatids segregate away from each other during mitosis. It is X segregation that is useful for genetic mosaic analysis because it produces clones of homozygous mutant daughter cells from heterozygous mothers. Our studies confirm the predictions made from studies in Drosophila that suggest that X segregation will not be limited to organisms with strong mitotic pairing, because the forces (sister-chromatid cohesion) responsible for X segregation are an elemental feature of mitosis in all eukaryotes. Our studies also show that genetic mosaic analysis in mice is feasible, at least for certain chromosomal regions.

Animals↗