Construction Of PCR～(?) 3.1/p16 Plasmid And Its Transgenic Research

The p16 protein was initially cloned as a protein associated with the cyclin D-dependent kinase CDK4 by the yeast two-hybrid protein interaction screen, and it was shown to be an efficient inhibitor of the CDK4 kinase activity. The pl6 gene (p16, MTS1, CDKN2) was localized to chromosome 9p21. Polymer-ase chain reaction and Southern blot analysis confirmed the frequent deletion or rearrangement of the CDK4-inhibitor gene in melanomas, gliomas, lung cancers and leukaemias, and the absence of detectable gene transcripts. Most published papers focused on modes of p16^INK4a inactivation in carcinogenesis and its possibility as potential anti-cancer drugs, but a little on transgenic study on the whole animal level. To construct p16 eukaryotic expression plasmid and produce transgenic mice carrying exogenous pl6 is more helpful to clarifying the role of p16^INK4a on the whole animal level.This paper consists of two parts. Construction of pCR(?)3. 1/p16 eukaryotic expression plasmid and establishment of NIH3T3 cell line with stable expression of exogenous p16 gene is described in the first part and production of transgenic mice carrying exogenous pl6 gene by microinjection in the second part.The First Part Construction of pCR? 3. l/pl6 Plasmid andEstablishment of NIH3T3 Cell Lines with StableExpression of Exogenous pl6 GenePurposeTo construct pCR(?)3. 1/p16 eukaryotic expression plasmid and to establish NIH3T3 cell line with stable expression of exogenous pl6 gene in order to lay afoundation for establishment of transgenic mice carrying exogenous p16 by micro-injection.Materials And MethodsHuman p16 cDNA was subcloned in to pCR?3. 1 and pCR?3. 1/p16 eu-karyotic expression plasmid was constructed. The recombinant plasmid was transferred in DH5α. Ecoli and the positive clone was identified by double digestion with EcoR I /Xho I , DNA sequencing and bioinformatics diagnosing . The identified pCR?3. 1/p16 was transfected in to NIH3T3 cell line with lipofectin. Ten G418-resistant colonies were obtained and two of them were confirmed as positive by reverse transcriptase-PCR (RT-PCR)ResultspCR?3. 1/p16 eukaryotic expression plasmid was obtained, p16 mRNA was expressed in those NIH3T3 cells transfected by pCR?3. 1/p16, the growth of which could be significantly suppressed by exogenous gene p16 cDNA.DiscussThe normal levels of expression of p16^INK4a are extremely low in most tissues. In mice, p16^INK4a expression is only detected in some adult tissues by RT PCR. An increase in the number of population doublings or the presence of on-cogene in a normal cell can activate the expression of p16^INK4a resulting in cell-cycle arrest and growth arrest in senescence. By microinjecting p16 protein directly in to human normal cell line, IMR-90, Lukas showed that wild-type p16 protein arrests normal diploid cells in late G1, whereas a tumour-associated mutant protein of p16 does not. Significantly, the ability of p16 protein to induce cell-cycle arrest is lost in cells lacking functional RB. In 1995, Koh used a plasmid microinjection assay in NIH3T3 cells, showing that microinjection of the p16 expression construct blocked progression in to S phase in a dose-dependentmanner and that co-injection of either cdk4 or cdk6 expression plasmids prevented pl6-mediated growth arrest. That the growth of NIH3T3 cells transfected by pCR 3. l/pl6 expression construct could be significantly suppressed by exogenous gene pl6 cDNA in this study showed no difference from the viewpoints a-bove.One of the most surprising discoveries of recent years has been the realization that the INK4a locus contains an overlapping gene named ARF and encoding p19^ARF ( also called p14^ARF when referring specifically to the human version). The INK4a and ARF genes have their own separate promoters, each of which produces a different transcript: the INK4a transcript is formed by exons 1 α, 2 and 3; and the ARF transcript is formed by exons 1 β, 2 and 3. Although exons 2 and 3 are common to INK4a and ARF transcripts, they are read in different frames and, consequently, the corresponding proteins do not share amino acid sequence homology. Engineered alterations of the INK4a/ARF locus have served to test the function of p16^INK4a and p19^ARF in tumor suppression. Two mutations have been introduced so far into the INK4a/ARF locus: elimination of exons 2 and 3 ( INK4a/ARF^â–³ex2,3) , and elimination of exon 1β (ARF^â–³1β). Both mutant mice, INK4a/ARF^â–³ex2,3 and ARF^â–³1β, have strikingly similar pheno-types, which suggest that p19^ARF is more relevant than p16^INK4a for tumor suppression in mice.Construction of pCR 3. 1/p16 eukaryotic expression plasmid and establishment of NIH3T3 cell line with stable expression of exogenous p16 gene would be able to lay a foundation for establishment of transgenic mice carrying exogenous p16 which is more helpful to clarifying the role of p16^INK4a on the whole animal level.Conclusion1. pCR(?)3. 1/p16 eukaryotic expression plasmid was constructed successfully.2. NIH3T3 cell line with stable expression of exogenous p16 gene was established successfully.3. The growth of NIH3T3 cells could be significantly suppressed by Expression vectors encoding human p16^INK4a.The Second part Production of transgenic mice carrying human p16^INK4a by microinjectionPurposeTo produce the transgenic mice carrying human p16^INK4a by microinjection.Materials And Methods204 female mice of 615 strain were induced to give superovulation. The fertilized eggs were harvested and microinjected with pCR 3. 1/p16 into their pro-nuclei. After microinjection, the survival and healthy fertilized eggs were implanted into the oviducts of pseudopregnant female of Kunming mice to let them develop until delivery. PCR analysis was used to examine the integration of transgene in the offspring's genomes.ResultsIn this experiment, 510 fertilized eggs were microinjected and 350 of them survived and were healthy after microinjection. The survival rate was 68. 6%. These eggs were implanted into the oviducts of 21 pseudopregnant female mice, of which 12 were pregnant and 46 offsprings were born. The produce rate of pregnant recipients was 22.4%. The total produce rate was 13%. None of 45 offsprings was confirmed as positive by PCR.DiscussThe microinjection of DNA directly into the pronuclei of fertilized zygotes isthe most extensively and successfully used method of gene transfer in the mouse. This method results in the stable chromosomal integration of the foreign DNA in 10 40% of the resulting mice. There were many reasons for no founder among the 45 pups born in this study. In general, the main reasons for low proportion of founders among the pups born are probably as follows: (1) the volume of injected DNA was too low; (2) the DNA concentration was lower than estimated; (3) the DNA was diluted by backflow of culture medium into the injection pipette; (4) the DNA was not injected in to the pronucleus because (a) the pronuclear membrane was not penetrated, resulting in DNA position in the cytoplasm or (b) neither the pronuclear membrane nor the oolemma was penetrated resulting in DNA position outside the zygote plasma membrane. (5) the vasectomized males were not correctly vasectomized, so that the foster mother carried her own fertilized embyos in addition to the injected zygotes; (6) the expression of the transgene is toxic to the developing fetus. In our study we have increased the degree of pronuclear swelling , remeasured the concentration of the injected DNA , squeezed on the plunger once before entering the injection pipette in to the zygote ,only transferred zygotes where a clear pronuclear swelling can be detected. Moreover Kunming mice were used both as vasectomized males and foster females and no albino mice are detected in the litters. So we could basicly eliminate the reasons mensioned above: (1)-(5). Seeing that expression vectors encoding human or mouse p16^INK4a caused G₁ phase arrest in NIH3T3 fibroblasts, we concluded that the expression of pCR?3. 1/p16 may suppress the development of transgenic embyos , resulting in no founder among the 45 pups born . If this observation is accurate, as I believe it is , the implications are far reaching. It may well be that in addition to a need for controlling transgene expression by developmental or cell-type specific regulatory elements, there should be another need for temporal/inducible regulation to produce the transgenic mice carrying human p16^INK4a.