β-2 Adrenergic receptor gene polymorphism and response to propranolol in cirrhosis

AIM: To evaluate the association of β-2 adrenergic receptor(β2-AR) gene polymorphism with response of variceal pressure to propranolol in cirrhosis.METHODS: Sixty-four non-related cirrhotic patients participated in this study and accepted variceal pressure measurement before and after propranolol administration. Polymorphism of the β 2-AR gene was determined by directly sequencing of the polymerase chain reaction products from the DNA samples that were prepared from the patients.RESULTS: The prevalence of Gly16-Glu/Gln27 and Arg16-Gln27 homozygotes, and compound heterozygotes was 29.7%, 10.9%, and 59.4%, respectively.Patients with cirrhosis with Gly16-Glu/Gln27 homozygotes had a greater decrease of variceal pressure after propranolol administration than those with Arg16-Gln27 homozygotes or with compound heterozygotes(22.4% ± 2.1%, 13.1% ± 2.7% and 12.5% ± 3.1%,respectively, P < 0.01).CONCLUSION: The variceal pressure response to propranolol was associated with polymorphism of β 2-AR gene. Patients with the Gly16-Glu/Gln27 homozygotes probably benefit from propranolol therapy.

Copy number and zygosity determination of transgenic rapeseed by droplet digital PCR

Establishing an accurate and rapid method for copy number and zygosity determination can accelerate genetic engineering research process. In this study, droplet digital PCR (ddPCR), an emerging DNA absolute quantification technology, was used to identify single-copy homozygous transgenic lines from a batch of T0 transgenic rapeseed harboring 11 exogenous elements. Copy number of exogenous gene was evaluated in T0 generation based on calculated ratio between transgene and reference CruA gene, single-copy transformants were selected for selfing followed by subsequent zygosity analysis. Single-copy homozygous transgenic plants were successfully screened out in T1 generation by ddPCR.Segregation analysis with T2 seedlings verified that identification results of ddPCR were accurate and reliable. This study provides a novel rapid and accurate method for copy number and zygosity determination in transgenic rapeseed which overcomes disadvantages of traditional Southern analysis and recently developed real-time quantitative method.

Transgenic Tobacco Plants With Efficient Insect Resistance

Insecticidal protein gene CryIA(c)from Bacillus thuringiensis HD-1(B.t.toxin gene)with 5’-end modified and 3’-end deleted to 4 different lengths were inserted downstream of 35S promoterwith double enhancer and"Ω’"fragment of TMV-RNA cDNA in the binary vector pBin438 to constructthe chimeric expression vector of B.t.toxin gene.Leave stripes of tobacco plant var.NC89 widelygrown in China were transformed with A.tumefaciens LBA4404 harbouring the above expression vectorsrespectively,and kanamycin resistant tobacco plants were regenerated.Insect test with tobacco budwormH.assulta showed that insect-resistant transform.ants could be obtained from the regenerated plantstransformed with B.t.genes of different lengths though highest percentage(～50%)of plants with ahigh morality(90%-100%)to the testing insects is among those transformed with 1.8-kb toxin gene.Genetic,molecular and biological analyses of T1 and T2 progenies of plants with high efficient insect re-sistance showed that B.t.toxin gene and the character of insect resistance have been inherited in the pro-genies.Insect-resistant homozygotes D8-14 and D19-8 have been selected for small-scale field tests.

A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system

Clustered regulatory interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 nuclease(Cas9),the third-generation genome editing tool,has been favored because of its high efficiency and clear system composition.In this technology,the introduced double-strand breaks(DSBs)are mainly repaired by non-homologous end joining(NHEJ)or homology-directed repair(HDR)pathways.The high-fidelity HDR pathway is used for genome modification,which can introduce artificially controllable insertions,deletions,or substitutions carried by the donor templates.Although high-level knock-out can be easily achieved by NHEJ,accurate HDR-mediated knock-in remains a technical challenge.In most circumstances,although both alleles are broken by endonucleases,only one can be repaired by HDR,and the other one is usually recombined by NHEJ.For gene function studies or disease model establishment,biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes.Thus,there is an urgent need for an efficient biallelic editing system.Here,we developed three pairs of integrated selection systems,where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker.Flanked by homologous arms containing the mutated sequences,the selection cassettes were integrated into the target site,mediated by CRISPR/Cas9-induced HDR.Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance.We tested this novel method on the amyloid precursor protein(APP)and presenilin 1(PSEN1)loci and demonstrated up to 82.0%biallelic editing efficiency after optimization.Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.