[Myocardial protection of del Nido cardioplegia in adult cardiac and major vascular surgery with long aortic cross-clamp time].

Objective: To explore the safety and myocardial protection efficacy of del Nido cardioplegia in adult cardiac and major vascular surgery with long aortic cross-clamp (ACC) time. Methods: A total of 2 536 patients who underwent adult cardiac and major vascular surgery with ACC time>90 min at Beijing Anzhen Hospital from March 2018 to March 2023 were collected. The patients were divided into two groups according to the type of cardioplegia solution: the del Nido cardioplegia solution group (DC group) and the cold blood cardioplegia solution group (BC group). Preoperative baseline data of the patients (age, gender, comorbidities, ejection fraction, etc) were adjusted using propensity score matching (PSM). Cardiopulmonary bypass (CPB) time, ACC time, total amount of cardioplegia solution, in-hospital mortality rate, length of intensive care unit (ICU) stay, mechanical ventilation time, postoperative complications, left ventricular ejection fraction, and troponin levels were compared between the two groups. Results: After PSM, a total of 306 patients were included, including 223 males and 83 females, with a mean age of (52.0±12.3) years. There were 153 cases in the DC group and 153 cases in the BC group. Compared with the DC group, the cross-clamp time was longer [109(100, 150) min vs 102(91, 133) min, P<0.001], the rate of return to spontaneous rhythm was lower [51.6% (79/153) vs 86.9%(133/153), P<0.001], and intraoperative peak glucose was higher [12.6 (6.5, 15.9) mmol/L vs 10.1 (8.5, 12.4) mmol/L, P=0.005] in the BC group. In addition, perioperative mortality [4.6% (7/153) vs 3.3% (5/153), P=0.132], stroke[3.9% (6/153) vs 3.3% (5/153), P=0.759], renal insufficiency [3.3% (5/153) vs 6.5% (10/153), P=0.186], atrial fibrillation [4.6% (7/153) vs 2.6% (4/153), P=0.652] and low cardiac output syndrome [3.9% (6/153) vs 4.6% (7/153), P=0.716] did not differ between the two groups. Compared with BC group, DC group had lower level of high sensitivity troponin (hsTnI) [1.2 (0.8, 1.8) µg/L vs 1.3 (0.9, 2.3) µg/L, P=0.030] and creatine kinase isoenzyme (CK-MB) [31.0 (20.0, 48.9) µg/L vs 37.0 (24.0, 58.9) µg/L, P=0.011] at 24 h postoperatively, and shorter length of ICU stay [35.6 (19.8, 60.5) h vs 42.6 (21.9, 83.6) h, P=0.015] and mechanical ventilation time [20.5 (15.5, 41.0) h vs 31.5 (17.1, 56.0) h, P=0.012]. Subgroup analysis showed that in the 120-180 minute subgroup, patients in the DC group had a shorter cross-clamp time [132 (124, 135) min vs 136 (124, 138) min, P<0.001], while levels of hsTnI [1.6 (1.1, 2.0) µg/L vs 1.4 (1.0, 2.6) µg/L, P=0.030] and CK-MB [38.8 (23.5, 55.5) µg/L vs 37.0 (24.5, 62.3) µg/L, P=0.011] were higher than those in the BC group. Conclusions: In adult cardiac and major vascular surgery with ACC times>90 min, comparable myocardial protection is observed with the use of DC compared with BC. Additional advantages in glycemic control, return to spontaneous rhythm, and improved surgical procedures make DN an attractive alternative for myocardial protection in adult cardiac surgery.

[Risk factors associated with in-hospital mortality in patients requiring extracorporeal membrane oxygenation in the perioperative period of heart transplantation].

Objective: To explore risk factors associated with in-hospital mortality in patients requiring extracorporeal membrane oxygenation (ECMO) in the perioperative period of heart transplantation. Methods: The data of ECMO cases in the perioperative period of heart transplantation from the Chinese Society of Extracorporeal Life Support (CSECLS) between January 2017 and December 2021 were retrospectively analyzed. These patients were divided into the survival group and non-survival group according to their outcomes at discharge. The demographics, indications and complications of ECMO between the two groups were compared, and the related risk factors of poor prognosis were analyzed. Results: A total of 77 patients were included in the study, including 67 males and 10 females, with a median age [M(Q1, Q3)] of 48 (36, 59) years. Sixty-three patients (81.8%) were successfully withdrawn from the ECMO and 46 patients (59.7%) survived to discharge. The median ECMO time was 139 (92, 253) hours. Compared with the survival group, the non-survival group (n=31) had more patients with chronic kidney disease before surgery [22.6% (7/31) vs 4.3% (2/46), P=0.034], and a higher proportion of continuous renal replacement therapy (CRRT) during ECMO [74.2% (23/31) vs 50.0% (23/46), P=0.034]. Moreover, the non-survival group had longer duration of extracorporeal circulation [262 (195, 312) vs 201 (155, 261) min, P=0.056] and higher lactate value in the first 24 hours of ECMO support [2.7 (2.1, 4.7) vs 2.3 (1.4, 3.8) mmol/L, P=0.060], but the differences were not statistically significant. Multivariate logistic regression analysis showed that perioperative application of CRRT was an independent risk factor for poor prognosis in ECMO patients during heart transplantation (OR=19.345, 95%CI: 1.209-309.440, P=0.036). Conclusion: CRRT treatment during ECMO is a risk factor for in-hospital mortality in patients undergoing heart transplantation.

[Clinical phenotype and gene mutation analysis of 12 patients with hereditary protein C deficiency in different families].

Objective: To investigate the molecular pathogenesis and clinical features of unrelated 12 patients with inherited coagulation protein C (PC) deficiency in Chinese population. Methods: The PC activity (PC:A) and PC antigen (PC:Ag) were detected by chromogenic substrate and enzyme linked immunosorbent assay, respectively. The nine exons and flanking sequences of the protein C (PROC) gene were amplified by polymerase chain reaction with direct sequencing, and the suspected mutations were validated by reverse sequencing (clone sequencing for deletion mutations) . Results: The PC:A of the 12 probands decreased significantly, ranging from 18% to 55%, and the PC:Ag of the 10 probands decreased significantly. Eleven mutations were found, out of which four mutations [c.383G>A (p.Gly128Asp) , c.997G>A (p.Ala291Thr) , c.1318C>T (p.Arg398Cys) , and c.532G>C (p.Leu278Pro) ] were discovered for the first time. Six mutations were in the serine protease domain, four mutations were located in epidermal growth factor (EGF) -like domains, and one mutation was located in activation peptide. There were two deletion mutations (p.Met364Trp fsX15 and p.Lys192del) , and the rest were missense mutations. Mutations p.Phe181Val and p.Arg189Trp were identified in three unrelated families. All mutations may be inherited, and consanguineous marriages were reported in two families. Among the probands, nine cases had venous thrombosis, two cases had poor pregnancy manifestations, and one case had purpura. Conclusion: Patients with PC deficiency caused by PROC gene defects are prone to venous thrombosis, especially when there are other thrombotic factors present at the same time.

[A homozygous variant in a consanguineous pedigree with inherited protein S deficiency].

Objective: To observe the clinical feature of familiar hereditary protein S deficiency, and to explore the related gene mutation. Methods: The blood samples were obtained from the proband and the family memebers(3 generations,6 persons). PROS1 gene of the proband and the family members was analyzed. The 15 exons and flanking sequence of PROS1 gene were analyzed by PCR and DNA sequencing. Results: Five out of 6 family members were diagnosed as having hereditary protein S deficiency. The proband suffered from pulmonary embolism. The others had no obvious thrombotic event. The gene sequencing revealed that the proband carried a c.-168C>T homozygous variant in the promoter of exon 1. His parents, brother and son all carried c.-168C>T heterozygosis variant at the same position. The gene of his wife was a wild type. Conclusion: A gene variant (c.-168C>T) of PROS1 was discovered in this Chinese family. Gene variant of PROS1 may result in protein S deficiency. Patients with protein S deficiency may suffer from vein thrombosis and(or) pulmonary embolism.

[Predictors of in-hospital mortality in adult postcardiotomy cardiacgenic shock patients successfully weaned from venoarterial extracorporeal membrane oxygenation].

Objective: To assess the factors associated with outcome of patients undergoing extracorporeal membrane oxygenation (ECMO) in a large ECMO center. Methods: Patients aged >18 years who received ECMO support for postcardiotomy cardiogenic shock were identified between January 2011 and December 2015. One hundred and seventy-seven patients (64.8%) successfully weaned from ECMO. These patients were divided into two groups depending on whether they could survive to hospital discharge: the survival group (group S, n=119) and death group (group D, n=58). Multivariate logistic regression was performed to identify risk factors independently associated with in-hospital mortality. Results: Compared to those from group D, patients in group S exhibited a younger age[(53.4±11.7) vs (58.9±11.5) years], a lower inotrope score at the beginning of ECMO [25(15, 60) vs 35.0(23, 60)], a lower average platelets transfusion [4.0(2.0, 5.2) vs 5.0(3.0, 7.2)U] (all P<0.05). There were shorter duration of ECMO support [95.0(73.0, 131.0) vs 120.0(95.8, 160.2) h], shorter ventilation time [137.0(70.0, 236.8) vs 215.0(164.0, 305.0) h], shorter stay in ICU [182.0(140.0, 236.0) vs 259.0(207.0, 382.0) h] and longer hospital stay after weaned from ECMO [14(11, 24) vs 8(4, 16) d] in group S patients compared to those in group D (all P<0.05). Age>65 years (P=0.046), neurologic complications (P<0.001) and lower extremity ischemia (P<0.001) during ECMO support, left ventricular ejection fraction<35% (P=0.011) and central venous pressure (CVP)>12 cmH(2)O(P=0.018) when weaned from ECMO, and the multi-organ function failure (P<0.001) after weaned from ECMO were independently associated with in-hospital mortality. Conclusions: Neurologic complications and lower extremity ischemia that occurred during ECMO, multi-organ function failure after weaned from ECMO had a significant impact on in-hospital mortality. Further studies are needed to prevent neurologic complications and lower extremity ischemia in these patients. Interventions that could reduce these complications may improve outcome.

Different impact of two mutations of a novel compound heterozygous protein C deficiency with late onset thrombosis.

We investigated the alteration of coagulation state in a protein C (PC) deficiency pedigree and the impact of the PC gene mutations. The pedigree of a proband with cerebral hemorrhagic infarction had sixteen members with four generations. The plasma levels of PC activity (PC:A), protein S activity (PS:A), factor V:C and factor VIII:C, and routine coagulation tests were measured. Nine exons of the PC gene (PROC) were sequenced. Plasma PC:A and PC antigen (PC:Ag) of the proband were 26 and 18%, respectively, which was significantly lower than normal ranges. Two heterozygous missense mutations of PC in the proband were identified, T>G at site 6128 (exon 7) and G>C at site 8478 (exon 9) resulting in F139V and D255H, respectively. The family members with F139V (N = 4) or D255H (N = 4) had lower levels of PC:A and PC:Ag than members with wild-type PROC (N = 6). D255H mutation caused a more significant decrease in the levels of PC:A, PC:Ag and factor V:C as compared to F139V mutation (P < 0.05). Two independent mutations, F139V and D255H, of PROC reduce PC function. Compound heterozygous condition of the two mutations can cause synergistic PC deficiency, but resulting in later onset of cerebral thrombosis.

Total blood volume of Asian patients undergoing cardiac surgery is far from that predicted by conventional methods.

AIM: Current cardiopulmonary bypass (CPB) procedures use non-hematic fluids to prime bypass circuits, often resulting in marked hemodilution. Patients' total blood volume (TBV) is estimated prior to hemodilution. We aimed to evaluate differences between calculation of TBV by Nadler's formula, a classic reference book method, and an established formula calculated by the authors. METHODS: A total of 285 patients of Asian origin received primary cardiac surgery between September 2010 and October 2011 in our institution. Patients' total blood volume was estimated by: 1) standard Nadler formula: TBV (men) =0.417H3+0.045TBM-0.030L TBM (total body mass, Kg); TBV (women) =0.414H3+0.0328 TBM-0.030L; 2) classic reference book method: patient's weight in kilograms times 7% (women) or 7.5% (men); and 3) our practical calculation: TBV=HCT2*(CPB prime volume + intravenous fluids before CPB - urine volume before CPB)/(HCT1- HCT2). RESULTS: Bland-Altman plotting revealed no mean differences between Nadler formula and reference book TBV measurements (Figure 1A). Differences in means (95% limit of agreement) for reference book/Nadler formulas was 0.52 (-0.21, 1.24, N.=285). Comparing authors' results with those of reference book/Nadler, TBV yielded divergent results. TBV correlated positively to patient's height (P=0.001) and body surface area (P<0.01), and correlated positively to height after controlling for age and gender (ß=87.3, SE=42.9, P=0.043). CONCLUSION: Total blood volume of Asian patients calculated by the authors differs markedly from that estimated by Nadler and classic reference book formulas, which suggests that more accurate calculation of TBV is needed for Asian cardiac patients requiring CPB, especially patients with valvular disease.

Identification of immunogenic proteins of Flavobacterium columnare by two-dimensional electrophoresis immunoblotting with antibacterial sera from grass carp, Ctenopharyngodon idella (Valenciennes).

Flavobacterium columnare is a Gram-negative bacterium causing columnaris disease of freshwater fish worldwide, and development of efficacious vaccines has been a continuous challenge in aquaculture. In this study, 14 proteins were identified from cellular components of F. columnare using an immunoblotting approach in two-dimensional electrophoresis map gels with antibacterial sera from grass carp, Ctenopharyngodon idella (Valenciennes), and then anti-grass carp-recombinant Ig (rIg) polyclonal antibodies. These proteins were characterized conclusively by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF/TOF MS). The 14 proteins are immunogenic molecules of F. columnare, including chaperonins DnaK, GroEL and trigger factor, and translation elongation factor G, translation elongation factor Tu, 30S ribosomal subunit protein S1, dihydrolipoamide succinyltransferase, succinyl-CoA synthetase, SpoOJ regulator protein, alcohol dehydrogenase, fructose-bisphosphate aldolase, 3-hydroxybutyryl-CoA dehydrogenase and two conserved hypothetical proteins. These identified immunogenic proteins may provide candidate molecules for the development of vaccines against columnaris disease.

Ontogeny of IgM-producing cells in the mandarin fish Siniperca chuatsi identified by in situ hybridisation.

The ontogeny of IgM-producing cells was studied in juvenile mandarin fish Siniperca chuatsi, an important fish in China's aquaculture sector. The IgM-producing cells were localised through in situ hybridisation with a probe complementary to the Ig mu-chain in lymphoid-related tissues, including head kidney, spleen, thymus, intestine and gills. In head kidney, transcripts of Ig mu were first detected at 20days post-hatching (dph) with a few positive signals, and the number of IgM-producing cells increased obviously from 39dph onwards. At 136dph, a large amount of positive cells were observed in the entire organ with clusters of these cells located around the blood vessels. In spleen, IgM-producing cells were found from 26dph onwards, followed by an increase until 67dph; clusters of positive cells were also detected around blood vessels at 102dph. In thymus, IgM-producing cells were first observed at 39dph; thereafter, no obvious increase was detected until 78dph. The positive cells in thymus were distributed mainly in the outer zone of thymus. A few IgM-producing cells were still observed in thymus of 1-year-old mandarin fish. IgM-producing cells were not detected in the intestine until 87dph, with several discrete positively stained cells distributed in the lamina propria. IgM-producing cells, scattered mainly in primary gill filaments around blood vessels, were detected in gills from 90dph. As in other teleosts, these results indicated that the head kidney appears to be the primary organ for IgM production in mandarin fish, and IgM-producing cells exist in all organs examined in the present study, implying their lymphoid role in fish. In addition, it is suggested that vaccination after 20dph may be much more effective in mandarin fish.

Gene structure of an antimicrobial peptide from mandarin fish, Siniperca chuatsi (Basilewsky), suggests that moronecidins and pleurocidins belong in one family: the piscidins.

The gene of piscidin, an antimicrobial peptide, has been cloned from the mandarin fish, Siniperca chuatsi. From the first transcription initiation site, the mandarin fish piscidin gene extends 1693 nucleotides to the end of the 3' untranslated region and contains four exons and three introns. A predicted 79-residue prepropeptide consists of three domains: a signal peptide (22 aa), a mature peptide (22 aa) and a C-terminal prodomain (35 aa). The shortage of XQQ motif in the prodomain of mandarin fish piscidin and the similar gene structure between moronecidins (piscidins) and pleurocidins may indicate that they are derived from the same ancestor gene. We thus suggest that piscidin should be used as a terminology for these antimicrobial peptides in the future. The mandarin fish piscidin mRNA was abundant in intestine, spleen, pronephros and kidney analysed by real-time polymerase chain reaction. After stimulation with lipopoly saccharides (LPS), a marked increase in transcripts was observed in most tissues, indicating that piscidin is not only a constitutively expressed molecule, but also has an increased response to bacterial infection. The synthetic, amidated mandarin fish piscidin exhibited different antimicrobial activity against different fish bacterial pathogens, especially against species of Aeromonas, which may to certain extent reflect the pathogenicity of these bacteria.

Characterization and expression analysis of TNF-related apoptosis inducing ligand (TRAIL) in grass carp Ctenopharyngodon idella.

TRAIL (Apo2 ligand) described as a type II transmembrane protein belonging to the TNF superfamily can induce apoptotic cell death in a variety of cell types. In the present study, a putative cDNA sequence encoding the 299 amino acids of TRAIL (GC-TRAIL) and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted GC-TRAIL sequence showed 44 and 41% identities to chicken and human TRAILs, respectively. In a domain search, a tumor necrosis factor homology domain (THD) was identified in the C-terminal portion of TRAILs. The GC-TRAIL gene consists of five exons, with four intervening introns, spaced over approximately 4 kb of genomic sequence. Analysis of GC-TRAIL promoter region revealed the presence of a number of putative transcription factor binding sites, such as Sp1, NF-kappaB, AP-1, GATA, NFAT, HNF, STAT, P53 and IRF1 sequences which are important for the expression of other TNF family members. Phylogenetic analysis placed GC-TRAIL and the putative zebrafish (Danio rerio) TRAIL obtained from searching the zebrafish database into one separate cluster near mammalian TRAIL genes, but apart from the reported zebrafish TRAIL-like protein, indicating that the GC-TRAIL is an authentic fish TRAIL. Expression analysis revealed that GC-TRAIL is expressed in many tissues, such as in gills, liver, trunk kidney, head kidney, intestine and spleen.

Characterization of two genes encoding leucine-rich repeat-containing proteins in grass carp Ctenopharyngodon idellus.

The cDNAs and genes of two different types of leucine-rich repeat-containing proteins from grass carp (Ctenopharyngodon idellus) were cloned. Homology search revealed that the two genes, designated as GC-GARP and GC-LRG, have 37% and 32% deduced amino-acid sequence similarities with human glycoprotein A repetitions predominant precursor (GARP) and leucine-rich alpha2-glycoprotein (LRG), respectively. The cDNAs of GC-GARP and GC-LRG encoded 664 and 339 amino acid residues, respectively. GC-GARP and GC-LRG contain many distinct structural and/or functional motifs of the leucine-rich repeat (LRR) subfamily, such as multiple conserved 11-residue segments with the consensus sequence LxxLxLxxN/CxL (x can be any amino acid). The genes GC-GARP and GC-LRG consist of two exons, with 4,782 bp and 2,119 bp in total length, respectively. The first exon of each gene contains a small 5'-untranslated region and partial open reading frame. The putative promoter region of GC-GARP was found to contain transcription factor binding sites for GATA-1, IRF4, Oct-1, IRF-7, IRF-1, AP1, GATA-box and NFAT, and the promoter region of GC-LRG for MYC-MAX, MEIS1, ISRE, IK3, HOXA9 and C/EBP alpha. Phylogenetic analysis showed that GC-GARP and mammalian GARPs were clustered into one branch, while GC-LRG and mammalian LRGs were in another branch. The GC-GARP gene was only detected in head kidney, and GC-LRG in the liver, spleen and heart in the copepod (Sinergasilus major)-infected grass carp, indicating the induction of gene expression by the parasite infection. The results obtained in the present study provide insight into the structure of fish LRR genes, and further study should be carried out to understand the importance of LRR proteins in host-pathogen interactions.

Characterization of two membrane-associated protease genes obtained from screening out-membrane protein genes of Flavobacterium columnare G4.

In order to identify genes encoding the outer membrane proteins (OMPs) of the myxobacter Flavobacterium columnare G(4), the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs. Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods. Two genes encoding a membrane-associated zinc metalloprotease and prolyl oligopeptidase are reported in this paper. The membrane-associated zinc metalloprotease gene (map) is 1800 bp in length, coding for 449 amino acids (aa). Despite the presence of a conserved motif HEXXH for all metalloproteases, the special HEXXH approximately 32 aa approximately E motif of the F. columnare G(4) Map and its low level of identity with other reported zinc-containing metalloproteases may imply that the membrane-associated zinc metalloprotease of F. columnare G(4) represents a new family of zincins. The gene encoding prolyl oligopeptidase (Pop), a serine proteinase, is 2352 bp in length, coding for 649 aa. Sequence homology analysis revealed that the Pop is also novel as it has < 50% identity with other reported prolyl oligopeptidase family proteins. The present study represents the first to employ anti-fish bacterial OMP sera to screen genes of membrane-associated proteases of fish pathogenic bacteria, and to provide necessary information for the examination of the role of the two genes in the infection and pathogenesis of F. columnare.

Spontaneous combined hyperlipidemia, coronary heart disease and decreased survival in Dahl salt-sensitive hypertensive rats transgenic for human cholesteryl ester transfer protein.

The acceleration of atherosclerosis by polygenic (essential) hypertension is well-characterized in humans; however, the lack of an animal model that simulates human disease hinders the elucidation of pathogenic mechanisms. We report here a transgenic atherosclerosis-polygenic hypertension model in Dahl salt-sensitive hypertensive rats that overexpress the human cholesteryl ester transfer protein (Tg[hCETP]DS). Male Tg[hCETP]DS rats fed regular rat chow showed age-dependent severe combined hyperlipidemia, atherosclerotic lesions, myocardial infarctions and decreased survival. These findings differ from various mouse atherosclerosis models, demonstrating the necessity of complex disease modeling in different species. The data demonstrate that cholesteryl ester transfer protein can be proatherogenic. The interaction of polygenic hypertension and hyperlipidemia in the pathogenesis of atherosclerosis in Tg[hCETP]DS rats substantiates epidemiological observations in humans.

The alpha1 Na,K-ATPase gene is a susceptibility hypertension gene in the Dahl salt-sensitiveHSD rat.

Despite the prevalence of essential hypertension, its underlying genetic basis has not been elucidated due to the complexities of its determinants. To identify a hypertension susceptibility gene, we used an approach that integrates molecular, transgenic, and genetic analysis using Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats ascertained for genotype and phenotype. To determine the role of the Dahl S Q276L alpha1 Na,K-ATPase gene variant, we developed transgenic Dahl S rats bearing the Dahl R wild-type (wt) alpha1 Na, K-ATPase cDNA directed by the cognate wt promoter region, Tg[wtalpha1]. Transgenic Dahl S rats exhibited less salt-sensitive hypertension, less hypertensive renal disease, and longer life span when compared with non-transgenic Dahl S controls. Total chromosome 2 linkage analysis of F2(SxR) male rats detects cosegregation of the alpha1 Na,K-ATPase locus with salt-sensitive hypertension. These data support the alpha1 Na,K-ATPase gene as a susceptibility gene for salt-sensitive hypertension in the Dahl S rat model, and provide the basis for the study of the alpha1 Na,K-ATPase locus in human hypertension.

Developmental regulation for collagen II gene expression in transgenic mice.

In order to evaluate the involvement of the type II collagen regulatory sequences in development, we have injected a construct containing a toxin gene under the control of the rat type II collagen promoter and enhancer. The construct, pDAS10-DTA, contained the diphtheria toxin A chain gene under the control of type II collagen sequences which had been used previously to target cartilagenous tissues in transgenics. Inspection of developing fetuses at various stages of gestation revealed a high number of aborted implants as well as abnormally developing fetuses. These abnormal fetuses were of small size, had shortened and underdeveloped limbs, cleft palates, and generally resembled a phenotype similar to chondrodystrophic mice. Histological comparisons of normal and abnormal fetuses indicated a reduced amount of extracellular matrix surrounding chondrocytes, and a disorganized appearance of the tissue. These results suggest that the expression of the toxin has occurred in chondrocytes and altered the survival and development of the transgenic mice. These results also indicate that the promoter and enhancer sequences contained in the transgene controlled the developmental expression of the type II collagen gene expression.

Differences in the efficiency and stability of gene expression after transfection and nuclear injection: a study with a chick delta-crystallin gene.

The efficiency of an exogenous gene's expression was compared after its transfection and injection into various mouse cells to systematically evaluate these two gene transfer techniques. Special attention was paid to the period of transient expression. The gene used was a derivative of chicken delta-crystallin gene with the 5' end region replaced by a promoter base sequence of a retrovirus. Nuclear injection was more efficient than transfection in several respects: it was roughly one thousand times more efficient in producing gene-expressing cells than the transfection technique; it produced positive cells in every challenged cell line in contrast to the results of some unsuccessful trials found with transfection; and the maximum expression of the exogenous gene in a gene-transferred cell was much higher after injection than after transfection. With the transfection technique, use of a DNA-calcium phosphate coprecipitate was slightly more efficient than the use of DEAE-dextran. The stability of gene expression in transfected and nuclear-injected cells differed greatly: Expression of the exogenous gene in transfected cells was transmitted to 92% of the daughter cells per division, whereas its expression in injected cells was transmitted to only 32% of the daughter cells. This great difference in stability probably reflects different states of the major fraction of the exogenous gene: integration into chromosomes in transfected cells versus extrachromosomal localization in injected cells.